JP2025527245A - Nucleic acid compositions containing amphiphilic oligoethylene glycol (OEG) conjugate compounds and methods of using such compounds and compositions - Google Patents

Abstract

The present invention relates generally to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligoethylene glycol (OEG) conjugate compounds (as an alternative to PEG lipids), and in particular to the use of such compositions for delivering nucleic acids to cells of a subject or in therapy, such amphiphilic OEG conjugate compounds, and conjugates of such amphiphilic OEG conjugate compounds.

Description

TECHNICAL FIELD The present invention relates generally to the field of nucleic acid (e.g., DNA or RNA, particularly mRNA) compositions comprising amphiphilic oligoethylene glycol (OEG) conjugate compounds (as an alternative to PEG lipids), and in particular to the use of such compositions for delivering nucleic acids to cells of a subject or in therapy, such amphiphilic OEG conjugate compounds, and conjugates of such amphiphilic OEG conjugate compounds.

BACKGROUND: The use of recombinant nucleic acids (e.g., DNA or RNA) to deliver foreign genetic information to target cells is well known. Recombinant nucleic acids can be administered to a subject in need thereof in naked form; however, recombinant nucleic acids are typically administered using compositions. For example, nucleic acids such as RNA can be delivered to a subject using a variety of delivery vehicles, most of which are based on cationic polymers or lipids that form nanoparticles with the nucleic acid. Nanoparticles are intended to protect nucleic acids such as RNA from degradation, enable delivery of nucleic acids such as RNA to target sites, and facilitate cellular uptake and processing by target cells. The efficiency of nucleic acid delivery can depend, in part, on the molecular composition of the nanoparticles, which can depend on a number of parameters, including particle size, formulation, and charge, or grafting with molecular moieties such as polyethylene glycol (PEG) or other ligands.

Grafting with PEG is thought to reduce serum interactions, increase serum stability, and extend circulation time, which may be beneficial for certain targeting approaches. Ligands that bind to receptors at target sites may be beneficial for improving targeting efficacy. Furthermore, PEGylation can be used in particle engineering. For example, when lipid nanoparticles (LNPs) are produced by mixing an aqueous phase of nucleic acid, such as RNA, with an organic phase of lipids, a certain fraction of the lipid mixture must be PEG-conjugated (such PEG-conjugated lipids contain at least 30 consecutive ethylene glycol repeat units); otherwise, the particles will aggregate during or after the mixing process. It has been shown that particle size can be controlled by varying the molar fraction of PEG-lipids containing various molar masses of PEG. Similarly, particle diameter can be controlled by varying the molar mass of the PEG moiety of the PEGylated lipid. Typical accessible sizes range from 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy-Nucleic Acids 1, e37). The particles thus formed have the added advantage of a long circulating half-life and minimal interaction with serum components, which is desirable for many drug delivery approaches, due to the PEG fraction. Without the PEG-lipid, discretely sized particles cannot form; they form large aggregates and precipitate. Therefore, one of the key roles of the PEG-lipid is to promote particle self-assembly by providing steric hindrance to the surface of nascent particles formed when nucleic acids, such as RNA, are rapidly mixed with a lipid-containing ethanol solution and bind to the nucleic acid. PEG steric hindrance prevents interparticle fusion and promotes the formation of a homogeneous population of LNPs, achievable with diameters <100 nm.

Despite these advantages, PEGylation of nanoparticles can also result in several effects that are detrimental to the intended drug delivery application. PEGylation of liposomes and LNPs is known to promote cellular uptake and endosomal escape, ultimately reducing overall transfection efficiency. In fact, the PEG shell provides steric hindrance to efficient binding of particles to cells and impedes endosomal release by preventing membrane fusion between liposome and endosomal membranes. This is why the type and amount of PEG-lipid used must always be carefully regulated: on the one hand, it must provide sufficient stealth effect for in vivo and stabilization aspects, and on the other hand, it must not interfere with transfection. This phenomenon is known as the "PEG dilemma."

In addition to reduced transfection efficiency, PEGylation is also associated with the accelerated blood clearance (ABC) phenomenon induced by anti-PEG antibodies and/or complement activation, as well as storage diseases (Bendele A et al., 1998, Toxicolocical Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; S.M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes in rats can significantly alter the pharmacokinetic behavior of a second dose when administered several days later (Laverman P et al., 2001, J. Pharmacol. Exp. Ther. 298(2), 607-12; Ishida et al., 2006, J. Control Release 115(3), 251-8). The phenomenon of "accelerated blood clearance" (ABC) is thought to be proportional to the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma induces rapid clearance of particles by the monophagocyte system (MPS), ultimately reducing drug efficacy.

Because PEG is widely used as an ingredient in foods, cosmetics, hygiene products, and pharmaceuticals, a certain percentage of the general population has "pre-existing" anti-PEG antibodies. Anti-PEG antibodies may be associated with reduced efficacy of PEGylated drugs and hypersensitivity reactions that can lead to severe allergic symptoms.

PEG can induce an immune response, requiring multiple injections, which should be avoided in certain applications. An example is nucleic acids (e.g., RNA, particularly mRNA) for protein replacement therapy. Here, the risk is particularly high due to the potential for intrinsic immunogenicity of nucleic acids (e.g., RNA). Another example is protein knockdown therapy using inhibitory RNA (e.g., siRNA), antisense oligonucleotides, or DNA-based therapy.

Therefore, there remains a need in the art for efficient compositions and methods for introducing nucleic acids, such as RNA, into cells that avoid the drawbacks associated with the use of PEG. The present invention addresses this and other needs.

The inventors have surprisingly discovered that the compositions, methods, polymer conjugate compounds (also referred to as amphiphilic OEG conjugate compounds of the present invention) and conjugates described herein fulfill the above-mentioned needs. In particular, the polymer conjugate compounds described herein have been shown to be resistant to binding by antibodies raised against the PEG structure and to be stable under physiological conditions. Polymer conjugate compounds and the polymer components of the polymer conjugate compounds and their conjugates can be synthesized by well-known procedures, such as solid-phase peptide synthesis (SPPS). Polymer conjugate compounds and conjugates can be end-group functionalized with various moieties for charge modulation or introduction of specific molecular moieties, such as ligands.

SUMMARY The present invention is defined by the appended claims.

In a first aspect, the present invention provides a composition comprising: (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer conjugate compound comprising: (a) a polymer comprising the following general formula (I): and (b) one or more hydrophobic chains:
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester, preferably an optionally substituted amide, an optionally substituted thioamide, or an ester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100.] This composition is also referred to as the nucleic acid composition of the present invention.

As demonstrated in this application, anti-PEG antibodies (polyclonal and IgG and IgM anti-PEG antibodies) raised against and binding to PEG (i.e., having at least 30 consecutive ethylene glycol repeating units) do not bind to polymers containing the structure of formula (I). Furthermore, the present invention demonstrates that polymers containing the structure of formula (I) are stable under physiological conditions.

In certain embodiments of this first aspect (particularly with respect to Formula (I)), ^X2 and ^X1 together are an optionally substituted amide. Thus, in certain embodiments, ^X1 is -C(O)- and ^X2 is ^-NR1- , where ^R1 is hydrogen or _C1-8 alkyl. In certain embodiments, ^X1 is ^-NR1- and ^X2 is -C(O)-, where ^R1 is hydrogen or _C1-8 alkyl.

In certain embodiments of this first aspect (particularly with respect to Formula (I)), ^X2 and ^X1 together are an optionally substituted thioamide. Thus, in certain embodiments, ^X1 is -C(S)- and ^X2 is ^-NR1- , where ^R1 is hydrogen or _C1-8 alkyl. In certain embodiments, ^X1 is ^-NR1- and ^X2 is -C(S)-, where ^R1 is hydrogen or _C1-8 alkyl.

In certain embodiments of this first aspect (particularly with respect to Formula (I)), ^X2 and ^X1 together are an ester. Thus, in certain embodiments, ^X1 is -C(O)- and ^X2 is -O. In certain embodiments, if ^X1 is -O-, then ^X2 is -C(O).

In certain embodiments of this first aspect (particularly with respect to Formula (I)), ^X2 and ^X1 together are a thioester. Thus, in certain embodiments, ^X1 is -C(S)- and ^X2 is -O-. In certain embodiments, if ^X1 is -O-, then ^X2 is -C(S)-. In certain embodiments, if ^X1 is -C(O)-, then ^X2 is -S-. In certain embodiments, if ^X1 is -S-, then ^X2 is -C(O)-.

In certain embodiments of this first aspect (particularly with respect to Formula (I)), X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl. For example, R ¹ can be hydrogen or methyl. In certain embodiments, R ¹ is hydrogen.

In certain embodiments of this first aspect (particularly with respect to formula (I)), Y is —CH ₂ — or —(CH ₂ ) ₂ —. In certain embodiments, Y is —CH ₂ —.

In certain embodiments of the first aspect, the polymer has the following general formula (II):
wherein R ¹ is hydrogen or C _1-8 alkyl.
In some embodiments of Formula (II), R ¹ is hydrogen or methyl. For example, R ¹ can be hydrogen. In some embodiments, R ¹ is the same in each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In some embodiments, R ¹ in at least one repeat unit is different from R ¹ in other repeat units (e.g., R ¹ is a specific alkyl (e.g., H) for at least one repeat unit and R ¹ is a specific alkyl (e.g., methyl) for at least one different repeat unit).

In some embodiments of this first aspect (particularly with respect to either of formulas (I) and (II)), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In certain embodiments of the first aspect, the polymer has the following general formula (III):
wherein R ¹ is hydrogen or C _1-8 alkyl.
In some embodiments of Formula (III), R ¹ is hydrogen or methyl. For example, R ¹ can be hydrogen. In some embodiments, R ¹ is the same in each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In some embodiments, R ¹ in at least one repeat unit is different from R ¹ in other repeat units (e.g., R ¹ is a certain alkyl (e.g., H) for at least one repeat unit and R ¹ is a certain alkyl (e.g., methyl) for at least one different repeat unit).

In certain embodiments of the first aspect, the polymer has the following general formula (IV):
Includes:

In certain embodiments of the first aspect, the polymer has the following general formula (IVa):
Includes.

In certain embodiments of this first aspect (particularly with respect to any of Formulas (I), (II), (III), (IV), and (IVa)), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In certain embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In certain embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 14. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, n is 12. In certain embodiments, n is 16.

In certain embodiments of this first aspect (particularly with respect to any of formulae (I), (II), (III), (IV) and (IVa)), the one or more hydrophobic chains are located at the ^X1 terminus or the ^X2 terminus of the polymer.

In some embodiments of this first aspect (particularly with respect to any of Formulas (I), (II), (III), (IV), and (IVa)), the one or more hydrophobic chains are independently selected from acyclic, preferably linear, hydrocarbyl groups, such as the hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the hydrocarbyl group has at least 8 carbon atoms, e.g., at least 10 carbon atoms, or at least 12 carbon atoms. The hydrocarbyl group may be saturated or unsaturated. If the polymer conjugate compound includes two or more hydrophobic chains, these chains may be the same or different. For example, if the polymer conjugate compound includes two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In some other embodiments, the two hydrophobic chains are different; for example, one may be saturated and the other (mono)unsaturated.

In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ^{21 .} , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and a 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and the 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²¹ is selected from the group consisting of C _1-6 alkyl and a 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and the 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
Includes.

In certain embodiments of formula (V) or (V'), ^X2 and ^X1 together are an optionally substituted amide. Thus, in certain embodiments, ^X1 is -C(O)- and ^X2 is ^-NR1- , where ^R1 is hydrogen or _C1-8 alkyl. In certain embodiments, ^X1 is ^-NR1- and ^X2 is -C(O)-, where ^R1 is hydrogen or _C1-8 alkyl.

In certain embodiments of formula (V) or (V'), ^X2 and ^X1 together are an optionally substituted thioamide. Thus, in certain embodiments, ^X1 is -C(S)- and ^X2 is ^-NR1- , where ^R1 is hydrogen or _C1-8 alkyl. In certain embodiments, ^X1 is ^-NR1- and ^X2 is -C(S)-, where ^R1 is hydrogen or _C1-8 alkyl.

In some embodiments of formula (V) or (V'), ^X2 and ^X1 together are an ester. Thus, in some embodiments, ^X1 is -C(O)- and ^X2 is -O. In some embodiments, if ^X1 is -O-, then ^X2 is -C(O).

In certain embodiments of formula (V) or (V'), ^X2 and ^X1 together are a thioester. Thus, in certain embodiments, ^X1 is -C(S)- and ^X2 is -O-. In certain embodiments, if ^X1 is -O-, then ^X2 is -C(S). In certain embodiments, if ^X1 is -C(O)-, then ^X2 is -S-. In certain embodiments, if ^X1 is -S-, then ^X2 is -C(O)-.

In certain embodiments of formula (V) or (V'), X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or C _1-8 alkyl. For example, R ¹ can be hydrogen or methyl. In certain embodiments, R ¹ is hydrogen.

In certain embodiments of formula (V) or (V'), Y is -CH ₂ - or -(CH ₂ ) ₂ -. In certain embodiments, Y is -CH ₂ -.

In some embodiments of formula (V) or (V'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments of Formula (V) or (V'), ^R1 is hydrogen or methyl. For example, ^R1 can be hydrogen. In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and a specific alkyl (e.g., methyl) for at ^least one different repeat unit).

In some embodiments of formula (V) or (V'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VI) or (VI'):
wherein z, n, ^R2 , and ^R3 are as defined for formulas (V) and (V'); and ^R1 is hydrogen or _C1-8 alkyl.
Includes.

In some embodiments of Formula (VI) or (VI'), ^R1 is hydrogen or methyl. For example, ^R1 can be hydrogen. In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and a specific alkyl (e.g., methyl) for at ^least one different repeat unit).

In some embodiments of formula (VI) or (VI'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments of formula (VI) or (VI'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VII) or (VII'):
wherein n, ^R2 , and ^R3 are as defined for formulas (V) and (V'); and ^R1 is hydrogen or _C1-8 alkyl.
Includes.

In some embodiments of Formula (VII) or (VII'), ^R1 is hydrogen or methyl. For example, ^R1 can be hydrogen. In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and a specific alkyl (e.g., methyl) for at ^least one different repeat unit).

In some embodiments of formula (VII) or (VII'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In certain embodiments of the first aspect, the polymer conjugate compound has the following general formula (VIII), (VIIIa), (VIII'), or (VIIIa'):
wherein n, ^R2 , and ^R3 are as defined for formulae (V) and (V').
Includes:

In some embodiments of formula (VIII), (VIIIa), (VIII'), or (VIIIa'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In certain embodiments of this first aspect (particularly with respect to any of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), ^R2 is ^R4 or ^-L1 ( ^R4 ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group; ^L1 is a linker; and p is 1 or 2.

In certain embodiments, ^L1 comprises at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or is attached to a divalent functionalized moiety at the terminal where the alkylene group is attached to ^R4 , wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocathion, thiocarbamate ... Carbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemi and/or each divalent functionalized moiety is independently selected from cetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate , thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

In some embodiments, L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ]-(C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(=N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N=)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; C _1-6 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁵ is selected from the group consisting of C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl optionally includes halogen, C _1-3 alkyl, —OH, —CN and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to another hydrophobic chain R ⁴ .

In certain embodiments, ^L1 further comprises at least one additional bifunctionalized moiety through which ^R2 is bonded ^{to X1} of formula (V) (or the carbonyl group of any of formulas (VI), (VII), (VIII), and (VIIIa)) or to ^X2 of formula (V') (or the N atom of any of formulas (VI'), (VII'), (VIII'), and (VIIIa')). In some embodiments, the at least one further difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidinium, thiomethyl ... dino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl and thiocarbonyl, wherein if ^L1 further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a _C1-6 -alkylene group.

In some embodiments, L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)O-P(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)O-P(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when _p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C is selected from the group consisting of _1-6 alkyl, aryl, aryl(C _1-6 alkyl) and a counter cation (e.g., the counter cation can be a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ .

In an embodiment, L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-OC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ ) _O (C _1-6 -alkylene)NH-, [*-NHC(O)] p (C _1-6 -alkylene)OP(O)(OR ²⁷ ) _O (C _1-6 -alkylene)OP(O)(OR ²⁷ ) _O (C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) ₂ N-, and [*-C(O)NH](C _1-6 -alkyltriyl)O-, or L ¹ is (*-)(R ²⁶ )N-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a countercation (e.g., the countercation can be a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ .

In certain embodiments of this first aspect (particularly with respect to any of formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)NH-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)—, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )—O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O-, (R ⁴ ) ₂ N- and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O- or R ² is (R ⁴ )(R ²⁶ )N-, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a countercation (e.g., the countercation can be a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ .

In certain embodiments of this first aspect (particularly with respect to any of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), ^R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety and a ceramide moiety, or ^R2 is a monoalkylamine moiety.

In some embodiments of this first aspect (particularly with respect to any of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII'), and (VIIIa')), each of the one or more hydrophobic chains (i.e., each ^R4 ) is independently an acyclic, preferably linear, hydrocarbyl group, such as the hydrophobic (e.g., lipophilic) chain of a natural lipid. In some embodiments, the one or more hydrocarbyl groups independently have at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The one or more hydrocarbyl groups may be saturated or unsaturated. If the polymer conjugate compound contains two or more hydrophobic chains, these chains may be the same or different. For example, if the polymer conjugate compound contains two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In certain other embodiments, the two hydrophobic chains are different, e.g., one may be saturated and the other (mono)unsaturated, and/or the two hydrophobic chains are different in length. Examples of the one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, e.g., hydrocarbyl chains of naturally occurring fatty acids, having at least 8 carbon atoms. In specific examples, the one or more hydrophobic chains include hydrocarbyl chains of caprylic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol, stearic alcohol, arachidyl alcohol, behenic alcohol, lignoceryl alcohol, cerotinoyl alcohol, oleyl alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

In certain embodiments of this first aspect (particularly with respect to any of Formulas (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), ^R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or ^R2 is a monomyristylamine moiety.

In certain embodiments of this first aspect (particularly with respect to any of formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -C(O)R ²¹ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²¹ is selected from the group consisting of C _1-6 alkyl and a 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide and a member of a targeting pair.

In certain embodiments of this first aspect (particularly with respect to any of formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R ³ is selected from the group consisting of H, C _1-3 alkyl, C _2-6 alkynyl, -C(O)R ²¹ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ and a member of a targeting pair, wherein the C _1-3 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² and is substituted with one or more substituents independently selected from the group consisting of C(O)R ²¹ and members of a targeting pair; R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ and members of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ and members of a targeting pair.

In certain embodiments of this first aspect (particularly with respect to any of formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl), —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ and a member of a targeting pair, wherein the C _1-3 alkyl group is optionally —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH ₃ , —C(O)NH(CH ₂ ) ₂ NH ₂ and a member of a targeting pair.

In certain embodiments of this first aspect (particularly with respect to any of Formulae (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII') and (VIIIa')), the targeting pair is selected from the following pairs: maleimide-thiol; thiol-alkyl halide (particularly brominated); azide-alkyne (particularly in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (particularly in Diels-Alder reactions); antigen-antibody specific for the antigen (including fragments or derivatives thereof); biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

In certain embodiments of the first aspect, the polymer conjugate compound has the formula:
wherein n is 5 to 25; R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl), and a member of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH ₃ , —C(O)NH(CH ₂ ) ₂ NH ₂ , and a member of a targeting pair; R ²⁷ is H or a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, for example, a quaternary ammonium or amine cation); and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
In some embodiments, n is 7 to 16, for example, 7 to 14 (preferably 8, 10, 12, 14, or 16); and/or ^R3 is H or —C(O)(C _1-3 alkyl), wherein the C _1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N ₃ , C _2-6 alkynyl, an antigen, and an antibody.

Further preferred embodiments of the polymer conjugate compound (particularly with respect to any of formulas (I), (II), (III), (IV), (IVa), (V), (V'), (VI), (VI'), (VII), (VII'), (VIII), (VIIIa), (VIII'), and (VIIIa')) are set forth under the heading "Polymer conjugate compound comprising: (a) a polymer comprising the structure of formula (I); and (b) one or more hydrophobic chains." In certain preferred embodiments of the first aspect, the polymer conjugate compound has any of formulas (V-1), (V-5), (V-17), and (V-25).

In some embodiments of the first aspect, the composition is substantially free of lipids or lipid-like substances comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeat units.

In some embodiments of the first aspect, the composition is also substantially free of other polymer-conjugated lipids. In some embodiments, the other polymer-conjugated lipids are polysarcosine-conjugated lipids and/or conjugates comprising a hydrophobic chain and a polyoxazoline (POX) and/or polyoxazine (POZ) polymer.

In certain embodiments of the first aspect, water is the major component of the composition and/or the total amount of solvents other than water in the composition is less than about 1.0% (v/v), e.g., less than about 0.5% (v/v). For example, the amount of water in the composition can be at least 50% (w/w), e.g., at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), at least 90% (w/w), or at least 95% (w/w). In particular, when the composition includes a cryoprotectant, the amount of water contained in the composition can be at least 50% (w/w), e.g., at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), or at least 90% (w/w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition can be at least 95% (w/w). Additionally or alternatively, the total amount of non-aqueous solvents in the composition may be less than about 0.5% (v/v), e.g., less than about 0.4% (v/v), less than about 0.3% (v/v), less than about 0.2% (v/v), less than about 0.1% (v/v), less than about 0.05% (v/v), less than about 0.01% (v/v), or less than about 0.005% (v/v). In this regard, a cryoprotectant that is liquid under normal conditions is considered a cryoprotectant, not a non-aqueous solvent. In other words, the optional limitation above that the total amount of non-aqueous solvents in the composition may be less than about 0.5% (v/v), e.g., less than about 0.4% (v/v), does not apply to a cryoprotectant that is liquid under normal conditions.

In some embodiments of the first aspect, the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg/L to about 500 mg/L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 1 mg/L to about 100 mg/L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 5 mg/L to about 500 mg/L, e.g., from about 10 mg/L to about 400 mg/L, from about 10 mg/L to about 300 mg/L, from about 10 mg/L to about 200 mg/L, from about 10 mg/L to about 150 mg/L, or from about 10 mg/L to about 100 mg/L, preferably from about 10 mg/L to about 140 mg/L, more preferably from about 20 mg/L to about 130 mg/L, and more preferably from about 30 mg/L to about 120 mg/L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from about 5 mg/L to about 150 mg/L, e.g., from about 10 mg/L to about 140 mg/L, from about 20 mg/L to about 130 mg/L, from about 25 mg/L to about 125 mg/L, from about 30 mg/L to about 120 mg/L, from about 35 mg/L to about 115 mg/L, from about 40 mg/L to about 110 mg/L, from about 45 mg/L to about 105 mg/L, or from about 50 mg/L to about 100 mg/L. In some embodiments, the concentration of nucleic acid (particularly RNA) in the composition is from 1 mg/L to about 50 mg/L or from about 10 mg/L to about 100 mg/L.

In some embodiments of the first aspect, the composition includes a cryoprotectant. In some embodiments of the first aspect, the composition is substantially free of a cryoprotectant.

In some embodiments of the first aspect, the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

In certain embodiments of the first aspect, the cationically ionizable lipid has the formula (X):
wherein L ¹⁰ , L ²⁰ , G ¹ , G ² , G ³ , R ³⁵ , R ³⁶ and R ³⁷ are as defined herein.
or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof. In some embodiments, the cationically ionizable lipid is selected from the following: Structures X-1 through X-36 (shown herein); or Structures A through G (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having Structure X-3. In some embodiments, the cationically ionizable lipid is DPL-14 (i.e., a lipid having Structure G).

In certain embodiments of the first aspect, the cationically ionizable lipid has formula (XI):
wherein R ₁ , R ₂ R ₃ , R ₄ , L ₂ , G ₂ and m are as defined herein.
In some embodiments, the cationically ionizable lipid is selected from structures (XIV-1), (XIV-2), and (XIV-3) (shown herein). In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-1. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-2. In some embodiments, the cationically ionizable lipid is a lipid having structure XIV-3.

In some embodiments of the first aspect, the cationic or cationically ionizable lipid is 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ... )-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or mixtures thereof.

In some embodiments of the first aspect, the cationically ionizable lipid is completely or partially replaced with a cationic lipid. In some embodiments, the cationic lipid is selected from structures XV-1 to XV-6 (shown herein).

In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises from about 20 mol % to about 80 mol %.

In certain embodiments of the first aspect, the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of a phospholipid and a steroid.

In one embodiment, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilaurate, and sphingomyelin. dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE).

In some embodiments, it comprises from about 5 mol% to about 30 mol% of the total lipids present in the phospholipid composition.

In some embodiments, the steroid comprises a sterol. In some preferred embodiments, the steroid comprises or is cholesterol.
contains or is cholesterol.

In some embodiments, the steroid comprises about 10 mol% to about 60 mol% of the total lipids present in the composition.

In some embodiments of the first aspect, the cationic or cationically ionizable lipid comprises about 20 mol% to about 70 mol% of the total lipid present in the composition; the polymer conjugate compound (amphiphilic OEG conjugate compound) comprises about 0.5 mol% to about 15 mol% (e.g., about 2 mol% to about 6 mol% or about 2 mol% to about 5 mol%) of the total lipid present in the composition; the phospholipid comprises about 5 mol% to about 25 mol% of the total lipid present in the composition; and the steroid comprises about 20 mol% to about 55 mol% of the total lipid present in the composition.

In some embodiments of the first aspect, the composition further comprises one or more additional lipids. For example, the one or more additional lipids can include a cationic lipid. In these embodiments, when a cationic lipid is present, the sum of the amount of (1) the cationically ionizable lipid and the amount of (2) the cationic lipid is used in the calculation. For example, if the amount of cationically ionizable lipid in the composition should be about 20 mol% to about 70 mol%, and the composition also contains a cationic lipid, the sum of the amount of (1) the cationically ionizable lipid and the amount of (2) the cationic lipid is about 20 mol% to about 70 mol%.

In certain embodiments of the first aspect, the lipids included in the composition are exclusively polymer conjugate compounds (i.e., amphiphilic OEG conjugate compounds) comprising a cationic or cationically ionizable lipid, a steroid, a neutral lipid, and a polymer of formula (I) as defined herein, particularly polymer conjugate compounds comprising a cationic or cationically ionizable lipid, a steroid, a phospholipid, and a polymer of formula (I) as defined herein.

In certain embodiments of the first aspect, the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationic or cationically ionizable lipid, and at least a portion of a polymer-conjugated compound comprising a polymer of Formula (I) as defined herein. In certain embodiments, the particles comprise or are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. In certain embodiments, the particles comprise or are LNPs. In certain embodiments, the particles comprise or are liposomes. In certain embodiments, the particles comprise or are LPXs. In certain embodiments, the particles comprise or are a mixture of LNPs and liposomes. In certain embodiments, the particles comprise or are a mixture of LNPs and LPXs. In certain embodiments, the particles comprise or are a mixture of liposomes and LPXs. In certain embodiments, the particles comprise or are a mixture of LNPs, liposomes, and LPXs.

In certain embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles comprise all of the lipids present in the composition (particularly the cationic or cationically ionizable lipids, if present, and all of the polymer conjugate compounds comprising one or more additional lipids and a polymer of formula (I) as defined herein).

In certain embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles comprise at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%) of the nucleic acid (especially RNA) present in the composition. In certain embodiments, the particles comprise at least 75%, preferably at least 85%, of the nucleic acid (especially RNA) present in the composition.

In some embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the aqueous phase is substantially free of nucleic acids.

In some embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the nucleic acid (e.g., RNA) is encapsulated within or associated with the particles.

In some embodiments of the first aspect, when the composition comprises particles dispersed in an aqueous phase, the particles have a size of about 30 nm to about 500 nm. In some embodiments, the particles have a size of about 50 nm to about 150 nm.

In some embodiments of the second aspect, the nucleic acid is DNA.

In some embodiments of the first aspect, the nucleic acid is RNA, preferably mRNA or inhibitory RNA (e.g., siRNA).

In certain embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and (i) contains modified nucleosides in place of uridine; (ii) has a codon-optimized coding sequence; and/or (iii) has a coding sequence with an increased G/C content compared to the wild-type coding sequence. In certain embodiments, the modified nucleosides are selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In some embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and comprises at least one or more of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In some embodiments, the RNA (e.g., mRNA) comprises all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. In some embodiments, the polyA sequence comprises at least 100 A nucleotides, wherein the polyA sequence is preferably a punctuated sequence of A nucleotides. In some embodiments, the 5' cap is a cap1 or cap2 structure.

In certain embodiments of the first aspect, the nucleic acid is RNA (e.g., mRNA) and encodes one or more polypeptides. In certain embodiments, the one or more polypeptides are pharmaceutically active polypeptides and/or comprise an epitope for inducing an immune response against an antigen in a subject.

In certain embodiments of the first aspect, the pharmaceutically active polypeptide and/or antigen or epitope is derived from or is a protein, immunogenic variant of the protein, or immunogenic fragment of the protein or immunogenic variant thereof of a pathogen. In certain embodiments, the pathogen is a pathogen that causes an infectious disease.

In certain embodiments of the first aspect, the nucleic acid is an inhibitory RNA (e.g., siRNA) that selectively hybridizes to and/or is specific for the target mRNA. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, particularly a pharmaceutically active peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with disease. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with cancer.

In a second aspect, the present invention relates to a method of delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect. It is understood that any embodiment described in the context of the first aspect also applies to any embodiment of the second aspect.

In a third aspect, the present invention relates to a method of delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described in the context of the first or second aspect also applies to any embodiment of the third aspect.

In a fourth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to a nucleic acid composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. It is understood that any embodiment described in the context of the first, second, or third aspects also applies to any embodiment of the fourth aspect.

In a fifth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid composition of the first aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to the nucleic acid composition of the first aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. It is understood that any embodiment described in the context of the first, second, third, or fourth aspect also applies to any embodiment of the fifth aspect.

In some embodiments of the second through fifth aspects, the subject is a mammal, such as a human.

In a sixth aspect, the present invention provides a polymer conjugate compound (also referred to herein as an amphiphilic OEG conjugate compound) comprising: (a) a polymer comprising a structure of formula (I); and (b) one or more hydrophobic chains. Preferred embodiments of the polymer conjugate compound of the sixth aspect are identified in the first aspect and are set forth herein under the heading "Polymer conjugate compound comprising: (a) a polymer comprising a structure of formula (I); and (b) one or more hydrophobic chains."

In a seventh aspect, the present invention provides a conjugate of (a) the polymer conjugate compound of the sixth aspect, which comprises a member of a targeting pair; and (b) a compound comprising another member of the targeting pair. In some embodiments, the compound comprising another member of the targeting pair further comprises a sugar, an amino acid, a peptide (e.g., an antigen or epitope), or an antibody. In some embodiments of the seventh aspect, the conjugate has the following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); each of m1 and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. In certain embodiments of the seventh aspect, the conjugate has one of the following formulas:
wherein n is 5 to 25, preferably 8, 10, 12, ₁₄ , or 16; in each case, --C(O) _{C.sub.17H.sub.35} refers to the moiety --C(O)( _CH.sub.2 ) _{.sub.16CH.sub.3} ( _stearoyl ); and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. In certain embodiments of the seventh aspect, the polymer conjugate compound comprising a member of a targeting pair has the following formula:
wherein n is 5 to 25, preferably 8, 10, 12, ₁₄ , or 16; in each case, --C(O) _{C.sub.17H.sub.35} refers to the moiety --C(O)( _CH.sub.2 ) _{.sub.16CH.sub.3} ( _stearoyl ); and Pept is an antigen or an antibody specific for said antigen.
the compound comprising the other member of the targeting pair is (i) an antibody specific for the antigen if Pept is the antigen; or (ii) a compound comprising the antigen if Pept is an antibody specific for the antigen; and the polymer conjugate compound comprising a member of the targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) the antibody specific for the antigen and (2) the antigen.

It is understood that any embodiment described in the context of the first, second, third, fourth, fifth or sixth aspect also applies to any embodiment of the seventh aspect.

In an eighth aspect, the present invention provides a composition comprising: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of the seventh aspect.

It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth or seventh aspect also applies to any embodiment of the eighth aspect.

In a ninth aspect, the present invention relates to a method of delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject a composition of the eighth aspect. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh or eighth aspect also applies to any embodiment of the ninth aspect.

In a tenth aspect, the present invention relates to a method of delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid encodes the therapeutic peptide or protein. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth or ninth aspect also applies to any embodiment of the tenth aspect.

In an eleventh aspect, the present invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein delivery of a nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the present invention relates to a composition of the eighth aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of a nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspect also applies to any embodiment of the eleventh aspect.

In a twelfth aspect, the invention relates to a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition of the eighth aspect, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the invention relates to a composition of the eighth aspect for use in a method of treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or eleventh aspects also applies to any embodiment of the twelfth aspect.

In a thirteenth aspect, the present invention provides a method of transfecting cells, comprising adding a composition of the first or eighth aspect to the cells; and incubating the mixture of the composition and the cells for a sufficient period of time. In certain embodiments, particularly where the nucleic acid is DNA or RNA (e.g., mRNA) and encodes a pharmaceutically active protein, the mixture of the composition and the cells is incubated for a sufficient period of time to allow expression of the pharmaceutically active protein. In certain embodiments, particularly where the nucleic acid is inhibitory RNA (e.g., siRNA) against a target mRNA, the mixture of the composition and the cells is incubated for a sufficient period of time to allow inhibition of transcription and/or translation of the target mRNA. In certain embodiments, the sufficient period of time is at least 1 hour (e.g., at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and/or up to about 48 hours (e.g., up to about 36 hours or up to about 24 hours).

In certain embodiments of the thirteenth aspect, the method is performed in vivo (i.e., the cells form part of an organ, tissue, and/or organism of a subject). In certain embodiments of the thirteenth aspect, the method is performed in vitro (i.e., the cells do not form part of an organ, tissue, and/or organism of a subject, e.g., the cells are in ex vivo cell culture).

It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh or twelfth aspect also applies to any embodiment of the thirteenth aspect.

In a fourteenth aspect, the present invention provides a pharmaceutical composition comprising the conjugate of the seventh aspect or the composition of the first or eighth aspect. In certain embodiments, the pharmaceutical composition further comprises one or more of a pharmaceutically acceptable carrier, diluent, and excipient. It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspects also applies to any embodiment of the fourteenth aspect.

In a further aspect, the present invention provides a kit comprising a composition of the first or eighth aspect, a polymer conjugate compound (amphiphilic OEG conjugate compound) of the sixth aspect, a conjugate of the seventh aspect, or a pharmaceutical composition described herein (e.g., a pharmaceutical composition of the fourteenth aspect). In some embodiments, the kit is for use in therapy, such as for inducing an immune response. In some embodiments, the kit is for use in inducing an immune response against a pathogen, such as for treating or preventing an infectious disease.

In a further aspect, the present invention provides a method for preparing an amphiphilic OEG conjugate compound, comprising the steps of: (a) providing an intermediate compound having formula (VII) or (VII') as disclosed herein, wherein for formula (VII), ^R2 is OH and ^R3 is H, acetyl, or Fmoc; and for formula (VII'), ^R2 is H, acetyl, or Fmoc and ^R3 is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, particularly a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety, thereby obtaining an amphiphilic OEG conjugate compound. A preferred embodiment of this aspect is shown under the heading "(a) a polymer comprising a structure of formula (I); and (b) a polymer conjugate compound comprising one or more hydrophobic chains." It is understood that any embodiment described in the context of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth or thirteenth aspects also applies to any embodiment of this aspect of the method of making an amphiphilic OEG conjugate compound.

Scheme of the anti-PEG ELISA assay used to determine the binding of anti-PEG polyclonal antibodies to various antigens (PEG, AEEA, pSAR). Biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin- _NH -AEEA14, biotin-capping-pSAR, or biotin- _NH -pSAR) were synthesized and captured on neutravidin-coated plates in wash buffer (1× PBS, 0.1% CHAPS) for 2 h at RT with gentle shaking. After washing with wash buffer (4x), the plates were incubated with various amounts of rabbit anti-PEG polyclonal serum (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (1x PBS, 0.1% CHAPS, 0.2% BSA) for 2 hours at room temperature with gentle shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (1:5000 dilution), and the plates were incubated for 1 hour at room temperature with gentle shaking. After washing with wash buffer (5x), HRP substrate was added, and the fluorescent signal was measured.

Figure 1. Anti-PEG ELISA assay results for various biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin-NH ₂ -AEEA14, biotin-capping-pSAR or biotin-NH ₂ -pSAR).

Results of further anti-PEG ELISA assays using monoclonal anti-PEG IgG (A) or monoclonal anti-PEG IgM (B) and various antigens (PEG36, capping-AEEA14, NH ₂ -AEEA14). PEG36 had a molecular weight of approximately 1.6 kDa. Capping-AEEA14 was Ac-AEEA14.

Stability of pAEEA under physiological conditions. Ac-AEEA14 (A) and NH ₂ -AEEA14 (B) were incubated in mouse or human plasma for 0 to 72 hours.

Particle size and PDI of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Particle size and PDI of the BM LNP formulation are also included.

Zeta potential of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Zeta potential of the BM formulation is also included.

Accessible RNA of DSPE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid, as determined by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of DSPE-AEEA14-AC containing LNPs with human whole blood using HY501 as the cationically ionizable lipid.

Viability of all test formulations after transfection with DSPE-AEEA14-AC, including LNPs using HY501 as the cationically ionizable lipid. Viability of BM_2 and other PEG-containing formulations is also included. Plotted is the mean (n=3) ± StDev.

Particle size and PDI of VE-AEEA14-AC, including LNPs using HY501 as the cationically ionizable lipid. Also included are particle size and PDI of the BM LNP formulation.

Zeta potential of VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. Zeta potential of the BM formulation is also included.

Accessible RNA of VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid, as determined by RiboGreen assay (A) or agarose gel electrophoresis (B).

Quantification of S1 protein expression after transfection with VE-AEEA14-AC containing LNPs using HY501 as the cationically ionizable lipid. (A) Variation of MFI (mean fluorescence intensity) versus VE-AEEA14-AC composition of the overall cell population. Data were fitted using a second-order polynomial function. (B) Viability of all test formulations. MFI and viability of BM_2 and other PEG-containing formulations are included. Plotted are mean values (n=3) ± StDev.

Yield and Purity of Peptide Intermediates. The peptide intermediates Ac-(AEEA) ₈ -OH (A–D) and Ac-(AEEA) ₁₄ -OH (E–H) were synthesized, and the yield and purity were determined using UPLC (A, B, E, F) and mass spectrometry (C, D, G, H). Samples for UPLC and mass spectrometry were taken immediately after cleavage from the resin, i.e., before the QC method (crude purity; Figure 15A, C, E, G) or after the QC method (final purity; Figure 15B, D, F, H).

Monitoring the synthesis of Ac-(AEEA)8-α-tocopherol. Reaction samples were taken and analyzed using the methods described herein. UPLC chromatograms are shown for samples taken after (A) 5 min and (B) the end of the reaction time (4 h).

Purity of amphiphilic compounds Ac-(AEEA) ₈ -α-tocopherol (A, B), Ac-(AEEA) 14 -α-tocopherol (C, D), Ac-(AEEA) ₁₄ -DMA (E, F), Ac-(AEEA) ₈ -DMG (G, H) and Ac-(AEEA) ₁₄ -DSPE (I, J) shown by exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J), _respectively .

Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid). Particle size and PDI of BM LNP formulations are also included.

Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid). Zeta potential of BM formulations is also included.

Accessible RNA of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid).

Quantification of S1 protein expression after transfection with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid). (A) Variation in overall cell population composition vs. MFI (mean fluorescence intensity). Data were fitted using a second-order polynomial function. (B) Viability of all test formulations. MFI and viability of BM_1 and BM_2 and other control formulations are included. Plotted are mean values (n=3) ± StDev.

Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

Accessible RNA of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 27A, B), mouse macrophage cell line (Raw) (Figure 27C, D), and liver cancer cell line (HepG2) (Figure 27E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng/well, 25 ng/well, and 50 ng/well of mRNA-loaded LNPs. Plotted are the mean values (n=3) ± StDev.

T cell targeting using LNPs with various stealth lipids and Alf lipids. LNPs were formulated with various lipid compositions (cargo: hy1.1 RNA/Luc RNA/Np proxy Venus 1:1:2 w/w; N/P ratio: 6; lipid mixture: HY501/cholesterol/DSPC/stealth lipid/Alf lipid. The lipid ratio was selected as [47.5/40.5/10/1.8/0.2] for the following combinations of stealth lipid and Alf lipid: C16 PEG2k ceramide/DSPE PEG2k Alfa, DSPE PEG2k/DSPE PEG2k. Alfa, DSPE-AEEA14/DSPE-AEEA14-Alfa, or VE-AEEA8/DSPE-AEEA14-Alfa. The lipid ratio was selected as [47.5/38.5/10/3.8/0.2] for the following combinations of stealth and Alf lipids: VE-PEG1k/DSPE-PEG2k-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. LNPs were loaded with aCD3 VHH x NbAlf ligand via post-functionalization [w/w* = ligand-to-cargo ratio 0.48]; RNA concentration: 0.1 μg/μl). The diameter of all LNPs was 100-170 nm, as determined by DLS measurements, with a PDI of less than 0.4.

For transfection studies, 10 μl (1000 ng dose) of each formulation was prediluted in 50 μl X-Vivo 15 medium in an ultra-low attachment 96-well plate. ¹⁰ thawed human PBMCs were diluted in 50 μl of 100% coagulation PHS and added to the nanoparticle dilutions. After 30 minutes of incubation (37°C, 5% _CO2 ), 30 μl of each transfection reaction was transferred to a second ultra-low attachment 96-well plate, and 170 μl of X-Vivo 15 medium + 100 U/ml IL2 was added per well. Cell dilutions were cultured for an additional 18 hours (37°C, 5% _CO2 ). Specific transfection (Thy1.1) was analyzed by flow cytometry in the following cell types: Per formulation condition tested, the percentage of transfected cells (CD2 negative cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells) within total transfected PBMCs (transfection, y-axis) is given.

Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

Zeta potential of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

Accessible RNA of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid).

In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 34A, B), mouse macrophage cell line (Raw) (Figure 34C, D), and liver cancer cell line (HepG2) (Figure 34E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng/well, 25 ng/well, and 50 ng/well of mRNA-loaded LNPs. Plotted is the mean value (n=3) ± StDev.

Particle size and PDI of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid).

Zeta potential of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as the cationically ionizable lipid).

Accessible RNA of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds and HY501 (as a cationically ionizable lipid).

In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic VE-(AEEA)n-AC conjugate compounds in a skeletal muscle cell line (C2C12) (Figure 40A, B), a mouse macrophage cell line (Raw) (Figure 40C, D), and a liver cancer cell line (HepG2) (Figure 40E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng/well, 25 ng/well, and 50 ng/well of mRNA-loaded LNPs. Plotted are mean values (n=3) ± StDev.

Particle size and PDIP of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as the cationically ionizable lipid).

Accessible RNA of LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing various amphiphilic OEG conjugate compounds and HY501 (as a cationically ionizable lipid).

In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing various amphiphilic OEG conjugate compounds in skeletal muscle cell line (C2C12) (Figure 45A, B), mouse macrophage cell line (Raw) (Figure 45C, D), and liver cancer cell line (HepG2) (Figure 45E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng/well, 25 ng/well, and 50 ng/well of mRNA-loaded LNPs. Plotted is the mean value (n=3) ± StDev.

Particle size and PDI of LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

Zeta potential of LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

Accessible RNA of LNPs containing amphiphilic OEG-conjugated compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) as measured by RiboGreen assay (A) or agarose gel electrophoresis (B).

Terminal complement complex (SC5b-9) formation after incubation of human serum with LNP formulations and controls. The horizontal dashed line indicates the level of SC5b-9 formation in PBS.

Hemolysis analysis after incubation (neutral pH conditions) of human whole blood with LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP).

In vitro expression (A, C, E) and viability (B, D, F) of LNPs containing amphiphilic OEG conjugate compounds and various cationic or cationically ionizable lipids (DODMA, DODAB, DOTMA, DOTAP) in a skeletal muscle cell line (C2C12) (Figure 51A, B), a mouse macrophage cell line (Raw) (Figure 51C, D), and a liver cancer cell line (HepG2) (Figure 51E, F). Firefly luciferase expression after 24 hours of incubation with 12.5 ng/well, 25 ng/well, and 50 ng/well of mRNA-loaded LNPs. Plotted are the mean values (n=3) ± StDev.

Functionalized LNPs prepared using five different OEG-conjugated compounds as stealth lipid particle sizes and PDIs.

Agarose gel electrophoresis of control, untreated functionalized LNPs (top row) and functionalized LNPs treated with release solution (bottom row).

Particle size and PDI of functionalized LNPs prepared with each OEG conjugate compound ((A) C14-pAEEA14-Ac, (B) DSPE-pAEEA14-Ac, (C) VitE-pAEEA14-Ac, (D) DMA-pAEEA14-Ac, and (E) VitE-pAEEA8-Ac) and subjected to three freeze-thaw cycles from −20°C to room temperature and −80°C to room temperature.

In vitro transfection with LNPs. A) Percentage of transfected cells (CD14+ monocytes, CD19+ B cells, CD4+ T cells, or CD8+ T cells) within total transfected PBMCs (transfection, y-axis) per test formulation. B) Total cell counts acquired by flow cytometry within 20 seconds. Counts are corrected for counting beads. BD FACS Lyric was used for sample acquisition. FlowJo was used for data analysis.

Ligand-mediated transfection of T cells in vivo. B6-hCD3EDG transgenic mice were injected intramuscularly with naked or LNP-formulated luciferase- and Thy1.1-encoding RNA mixtures (1:1 weight-to-weight mixture) (1 μg RNA dose/injected side; 2 μg total RNA dose/mouse). Analysis was performed 18 hours after injection. (A) Drainage analyzed via ex vivo bioluminescence imaging of popliteal, inguinal, axillary, and brachial lymph nodes and the spleen. (B) Cell-type-specific transfection analyzed by flow cytometry via detection of delivered Thy1.1 RNA expression in immune cell subtypes within popliteal and inguinal lymph nodes and the spleen. (C) T cell activation status analyzed by mean fluorescence intensity of CD69 surface expression within the indicated organs (after staining with anti-CD69-APC antibody). LN, lymph node; LNP, lipid nanoparticle; NK, natural killer; PMN, polymorphonuclear cell.

SEQUENCE DESCRIPTIONS The following table provides a list of certain sequences cited herein.

DETAILED DESCRIPTION The present invention is described in further detail below, but it should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Below, elements of the present invention are described in more detail. While these elements are listed with specific embodiments, it should be understood that they may be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only those embodiments explicitly described. The description is understood to support and encompass embodiments that combine any number of the disclosed and/or preferred elements with the explicitly described embodiments. Furthermore, any permutation and combination of all elements described herein is intended to be disclosed by the description of this application, unless the context indicates otherwise.

Preferably, the terms used herein are those described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)," H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

The practice of the present invention employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, as described in the literature of the art (e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser/Heathcook, "Organische Chemie", VCH, 1990; Beyer/Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey/Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Römpp Chemie Lexikon, Falbe/Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A Laboratory Manual, 2nd Edition, J. (See Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise contradicted by context. Any and all examples or exemplary language (e.g., "etc.") provided herein are intended merely to better illustrate the invention and are not intended to impose limitations on the scope of the invention as otherwise claimed. No language herein should be construed as indicating any non-claimed element essential to the practice of the invention.

Recitation of ranges of values herein is intended merely to serve as a shorthand method of referring to each separate value falling within that range. Unless otherwise specified herein, each separate value is included herein to the same extent as if it were individually set forth herein.

Several documents are cited by letter throughout this specification. Each document cited herein (including all patents, patent applications, scientific articles, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

DEFINITIONS The following definitions apply to all aspects of the present invention: The following terms have the following meanings unless otherwise specified: Any undefined term has its art-recognized meaning.

Throughout this specification and the claims that follow, unless the context requires otherwise, the terms "comprise" and variations thereof, such as "comprises" and "comprising," are understood to imply the inclusion of a stated member, integer, or step or group of members, integers, or steps, but not the exclusion of any other member, integer, or step or group of members, integers, or steps. The term "consisting essentially of" means the exclusion of any other member, integer, or step in any essential sense. The term "comprises" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each instance herein, the term "comprising" can be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each instance herein, the term "consisting essentially of" can be replaced with the term "consisting of."

When used in the context of describing the present invention, singular terms and similar expressions (particularly in the context of the claims) should be construed to include both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context.

As used herein, "and/or" shall be construed as a specific disclosure of each of the two specified properties or components, with or without the other. For example, "X and/or Y" shall be construed as a specific disclosure of (i) X, (ii) Y, and (iii) each of X and Y, to the same extent as if each were individually set forth herein.

In the present invention, the term "about" refers to an interval of accuracy that a person skilled in the art would understand to still estimate the technical effect of the property in question. This term typically indicates a deviation of ±5% from the indicated numerical value, e.g., ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and e.g., ±0.01%. As will be recognized by those skilled in the art, the specific deviation of the numerical value for a given technical effect depends on the nature of the technical effect. For example, natural or biological technical effects generally have larger deviations than man-made or engineered technical effects.

As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar terms includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

As used herein, "enhancement" and "increase" refer to the ability to cause an overall increase or enhancement in levels, e.g., by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more. In certain embodiments, these terms refer to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

As used herein, "physiological pH" refers to a pH of about 7.5 or about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

As used herein, "physiological conditions" refers to the conditions (especially pH and temperature) present in a living subject, particularly a human. Preferably, physiological conditions refer to physiological pH and/or a temperature of about 37°C.

As used herein, "% (w/v)" (or "% w/v") refers to weight/volume percent, which is a unit of concentration of the amount of solute measured in grams (g) expressed as a percentage of the total volume of the solution in milliliters (ml).

As used herein, "volume percent" or "%(v/v)" (or "%v/v") refers to volume percent, a unit of concentration of a liquid substance measured in milliliters (ml), expressed as a percentage of the total volume of the solution in milliliters (ml).

As used herein, "weight percent" or "% (w/w)" (or "% w/w") refers to weight percent, which is a unit of concentration of an amount of a substance measured in grams (g), expressed as a percentage of the total weight of the total composition in grams (g).

As used herein, "mol %" is defined as the ratio of the number of moles of a component to the total number of moles of all components multiplied by 100.

As used herein, "mol % of total lipid" is defined as the ratio of the number of moles of a lipid component to the total number of moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like substances.

The term "ionic strength" refers to the mathematical relationship between the number of different ionic species and their respective charges in a particular solution. Thus, ionic strength, I, is determined by the formula:
where c is the molar concentration of a particular ionic species and z is the absolute value of its charge.
The sum Σ is taken over all the different types of ions (i) in the solution.

According to the present invention, the term "ionic strength" refers, in some embodiments, to the presence of monovalent ions.

With regard to the presence of divalent inorganic ions, particularly divalent inorganic cations, their concentration or effective concentration (presence of free ions) in the presence of a chelating agent is, in some embodiments, sufficiently low to prevent RNA degradation. In some embodiments, the concentration or effective concentration of divalent inorganic ions is below the level that catalyzes the hydrolysis of phosphodiester bonds between RNA nucleotides. In some embodiments, the concentration of free divalent inorganic ions is 20 μM or less. In some embodiments, there is no or essentially no free divalent inorganic ions.

A "monovalent" compound refers to a compound that has only one functional group of interest. For example, a monovalent anion refers to a compound that has only one negatively charged group, preferably under physiological conditions.

A "divalent" or "dibasic" compound refers to a compound that has two functional groups of interest. For example, a dibasic organic acid has two acid groups. An example of a divalent cation is ^Ca.

A "polyhydric" or "polybasic" compound refers to a compound that has three or more functional groups of interest. For example, a polybasic organic acid has three or more acid groups.

"Monovalent moiety" refers to a monoradical, i.e., a moiety having a valence of one. Typical monovalent moieties include alkyl, alkenyl, aryl, etc.

A "divalent moiety" or "divalent moiety" refers to a diradical, i.e., a moiety having a valence of two. Typical divalent moieties include alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, etc. A further example of a divalent moiety is the C _1-6 -alkylene moiety in the group [*-S] _p (C _1-6 -alkylene)- if p is 1 (giving a group *-S(C _1-6 -alkylene)-, e.g. *-S-(CH ₂ ) ₆ - or *-S-CH ₂ - (where * represents the point of attachment to R ⁴ )).

A "polyvalent moiety" refers to a polyradical, i.e., a moiety having a valence of at least 3. For example, a "trivalent moiety" refers to a triradical, i.e., a moiety having a valence of 3. For example, by further removing an H atom from an alkylene group, the resulting alkylene is trivalent. A further example of a trivalent moiety is the C _1-6 -alkylene moiety in the group [*-S] _p (C _1-6 -alkylene)- if p is 2 (giving a group [*-S] ₂ (C _1-6 -alkylene)-, e.g. *-S-CH(S-*)(CH ₂ ) ₅ - or *-S-CH(S-*)(CH ₂ )- (where * represents the point of attachment to R ⁴ )). Another example of a trivalent moiety is the C _1-6 -alkyltriyl moiety in the group [*-C(O)NH](C _1-6 -alkyltriyl)-, where the [*-C(O)NH] moiety and the further hydrophobic chain are attached to the C _1-6 -alkyltriyl moiety, which is then attached to X ¹ (for formula (V)) or X ² (for formula (V')), either directly or via at least one further bifunctionalized moiety. Thus, the [*-C(O)NH](C _1-6 -alkyltriyl) moiety as an example of L ¹ , where the C _1-6 -alkyltriyl is attached directly to the other hydrophobic chain R ⁴ , may comprise at least the following structure:
[During the ceremony,
represents a bond in which C _1-6 -alkyltriyl is bonded to [*—C(O)NH];
and the other represents a bond connecting the C _1-6 -alkyltriyl to the remainder of the compound (e.g., a compound of formula (V)) (directly or via a further bifunctionalized moiety). When the C _1-6 -alkyltriyl is also substituted with one —OH moiety, at least the following structures are encompassed:

As used herein, "molar ratio" refers to the ratio in molar amounts between any two substances. For example, if a first substance is present in a composition in a 1 millimole (mmol) amount and a second substance is present in a composition in a 2 millimolar (mmol) amount, the molar ratio of the first substance to the second substance is 1:2, or 0.5.

"Osmolality" refers to the concentration of a particular solute expressed as osmoles per kilogram of solute in a solvent.

The term "lyophilize" or "freeze-drying" refers to freeze-drying a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or less than 1 Pa) to allow the freezing medium in the substance to sublimate directly from the solid phase to the gas phase. Thus, the terms "lyophilization" and "freeze-drying" are used interchangeably herein.

The term "spray drying" refers to the spray drying of a material by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer) where the solvent from the formed droplets evaporates, resulting in a dry powder.

The term "reconstitution" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

The term "freezing" refers to the solidification of a liquid, usually by the removal of heat. In some embodiments, freezing is the reverse of thawing.

The term "thawing" refers to the liquefaction of a solid, usually by the application of heat. In some embodiments, thawing is the reverse of freezing.

As used herein, the term "aqueous phase" with respect to compositions/formulations containing particles, particularly LNPs, liposomes, and/or lipoplexes, refers to the mobile or liquid phase, i.e., the continuous aqueous phase, that includes all components dissolved therein but (morphologically) excludes the particles. Thus, if particles such as LNPs are dispersed in an aqueous phase, and the aqueous phase is substantially free of compound X, the aqueous phase is virtually and practically free of X in a manner that is practically feasible; for example, the concentration of compound X in the aqueous composition is less than 1% by weight. However, it is also possible that particles dispersed in the aqueous phase may contain compound X in an amount greater than 1% by weight.

The term "recombinant" in the context of the present invention means "produced through genetic engineering." In certain embodiments, the "recombinant" in the context of the present invention does not occur in nature.

As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), is isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "naturally occurring" means "occurring in nature" and includes known entities as well as entities that have not been discovered and/or isolated in nature, but may be discovered and/or isolated from nature in the future.

As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and include temperatures of at least about 15°C, preferably about 15°C to about 35°C, about 15°C to about 30°C, about 15°C to about 25°C, or about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

The term "alkyl" refers to a monoradical of a saturated straight-chain or branched hydrocarbon. Preferably, an alkyl group contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms, abbreviated as C _1-12 alkyl (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, abbreviated as C _1-10 alkyl), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, iso-propyl (also known as 2-propyl or 1-methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2-dimethyl-propyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, and the like. A "substituted alkyl" is an alkylene group in which one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein. Examples of substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

The term "alkylene" refers to a diradical of a saturated straight chain or branched hydrocarbon. Preferably, the alkylene contains 1 to 12 (e.g., 1 to 10) carbon atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 1 to 8 carbon atoms, e.g., 1 to 6 or 1 to 4 carbon atoms. Examples of alkylene groups include methylene, ethylene (i.e., 1,1-ethylene, 1,2-ethylene), propylene (i.e., 1,1-propylene, 1,2-propylene (-CH(CH 3 )CH 2 -), 2,2-propylene (-C(CH ₃ ) ₂ -), and 2,2-propylene (-C(CH ₃ ) _{2 -).} -) and 1,3-propylene), butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3-butylene, 2,3-butylene (cis or trans or mixtures thereof), 1,4-butylene, 1,1-isobutylene, 1,2-isobutylene, and 1,3-isobutylene), pentylene isomers (e.g., 1,1-pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-isopentylene, 1,1-sec-pentyl, 1,1-neo-pentyl), hexylene isomers (e.g., 1,1-hexylene, 1,2 isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-hexylene, 1,6-hexylene, and 1,1-isohexylene), heptylene isomers (e.g., 1,1-heptylene, 1,2-heptylene, 1,3-heptylene, 1,4-heptylene, 1,5-heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1-isoheptylene), octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5-octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1-isooctylene), and the like. A straight chain alkylene moiety having at least three carbon atoms and a free valence at each end may also be designated by the number of methylenes (e.g., 1,4-butylene may also be referred to as tetramethylene). Generally, instead of using the terminal "ylene" for the alkylene moieties specified above, a terminal "diyl" may also be used (e.g., 1,2-butylene may also be referred to as butane-1,2-diyl). "Substituted alkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

The term "alkenyl" refers to a monoradical of an unsaturated straight-chain or branched hydrocarbon having at least one carbon-carbon double bond. In general, the maximum number of carbon-carbon double bonds in an alkenyl group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenyl group by 2, or, if the alkenyl group has an odd number of carbon atoms, rounding the result down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, an alkenyl group contains 2 to 12 (e.g. 2 to 10) carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, for example 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, an alkenyl group contains 2 to 12 (abbreviated as _C2-12 alkenyl) (e.g., 2 to 10) carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, for example, 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the cis (Z) or trans (E) configuration. Examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl (i.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, and 5-nonenyl. , 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. If an alkenyl group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkenyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

The term "alkynyl" refers to a straight- or branched-chain monovalent hydrocarbon moiety having at least one carbon-carbon triple bond and a total of 6 to 30, typically 6 to 20, and often 6 to 18 carbon atoms. An alkynyl group may optionally have one or more carbon-carbon double bonds. Generally, the maximum number of carbon-carbon triple bonds in an alkynyl group is equal to the number of carbon atoms in the alkynyl group divided by two; if the alkynyl group has an odd number of carbon atoms, the result is rounded down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, an alkynyl group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2, carbon-carbon triple bonds. "Substituted alkynyl" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of an alkynyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

The term "alkenylene" refers to an unsaturated straight-chain or branched hydrocarbon diradical having at least one carbon-carbon double bond. Generally, the maximum number of carbon-carbon double bonds in an alkenylene group can be equal to the integer obtained by dividing the number of carbon atoms in the alkenylene group by 2; if the alkenylene group has an odd number of carbon atoms, the result of the division is rounded down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. Preferably, the alkenylene group contains 2 to 12 (e.g. 2 to 10) carbon atoms, i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms), more preferably 2 to 8 carbon atoms, for example 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in preferred embodiments, an alkenylene group contains 2 to 12 (e.g., 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, for example, 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds, or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in the cis (Z) or trans (E) configuration. Examples of alkenylene groups include ethene-1,2-diyl, vinylidene (also known as ethenylidene), 1-propene-1,2-diyl, 1-propene-1,3-diyl, 1-propene-2,3-diyl, allylidene, 1-butene-1,2-diyl, 1-butene-1,3-diyl, 1-butene-1,4-diyl, 1-butene-2,3-diyl, 1-butene-2,4-diyl, 1-butene-3,4-diyl, 2-butene-1,2-diyl, 2-butene-1,3-diyl, 2-butene-1,4-diyl, 2-butene-2,3-diyl, 2-butene-2,4-diyl, 2-butene-3,4-diyl, and the like. If the alkenylene group is attached to a nitrogen atom, the double bond cannot be alpha to the nitrogen atom. "Substituted alkenylene" means that one or more hydrogen atoms (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the alkenylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

The term "cycloalkyl" refers to cyclic, non-aromatic versions of "alkyl" and "alkenyl," preferably having 3 to 14 carbon atoms, e.g., 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, cyclononyl, cyclononenyl, cyclodecyl, cyclodecenyl, and adamantyl. A cycloalkyl group can consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). "Substituted cycloalkyl" means that one or more hydrogen atoms (e.g., from one up to the maximum number of hydrogen atoms attached to the cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the cycloalkyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

The term "cycloalkylene" refers to a cyclic, non-aromatic version of "alkylene," and is a geminal, vicinal, or isolated diradical. In certain embodiments, the cycloalkylene is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and/or (ii) 3 to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 3-12-, or 3-10-membered). In certain embodiments, the cycloalkylene is a mono-, bi-, or tricyclic 3- to 14-membered (i.e., 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 3-12-, or 3-10-membered) cycloalkylene. Generally, instead of using the terminal "ylene" for the cycloalkylene moieties identified above, the terminal "diyl" can be used (e.g., 1,2-cyclopropylene can also be referred to as cyclopropane-1,2-diyl). Exemplary cycloalkylene groups include cyclohexylene, cycloheptylene, cyclopropylene, cyclobutylene, cyclopentylene, cyclooctylene, bicyclo[3.2.1]octylene, bicyclo[3.2.2]nonylene, and adamantanylene (e.g., tricyclo[3.3.1.1 ^3,7 ]decane-2,2-diyl). "Substituted cycloalkylene" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the cycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of an alkylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents as specified herein.

The term "cycloalkenylene" refers to the cyclic, non-aromatic version of "alkenylene" and may be a geminal, vicinal, or isolated diradical. Generally, the maximum number of carbon-carbon double bonds in a cycloalkenylene group can be equal to the integer obtained by dividing the number of carbon atoms in the cycloalkenylene group by 2; if the cycloalkenylene group has an odd number of carbon atoms, the result of the division is rounded down to the next integer. For example, for a cycloalkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the cycloalkenylene group has 1 to 6 (e.g., 1 to 4), i.e., 1, 2, 3, 4, 5, or 6 carbon-carbon double bonds. In certain embodiments, the cycloalkenylene is (i) monocyclic or polycyclic (e.g., bi- or tricyclic) and/or (ii) 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3 to 12 or 3 to 10 members). In certain embodiments, the cycloalkenylene is a mono-, bi-, or tricyclic 3 to 14-membered (i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14-membered, e.g., 3 to 12 or 3 to 10 members) cycloalkenylene. Exemplary cycloalkenylene groups include cyclohexenylene, cycloheptenylene, cyclopropenylene, cyclobutenylene, cyclopentenylene, and cyclooctenylene. "Substituted cycloalkenylene" means that one or more hydrogen atoms (e.g., from 1 up to the maximum number of hydrogen atoms attached to the cycloalkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the cycloalkenylene group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents specified herein).

The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. Preferably, an aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, e.g., 5, 6, or 10) carbon atoms, which can be arranged in a single ring (e.g., phenyl) or two or more fused rings (e.g., naphthyl). Examples of aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not include fullerene. "Substituted aryl" means that one or more hydrogen atoms (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) of the aryl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents specified herein. Examples of substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6-methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (i.e., 2-, 3-, or 4-methoxyphenyl).

The term "heteroaryl" or "heteroaromatic ring" refers to an aryl group, as defined above, in which one or more carbon atoms of the aryl group are replaced with an O, S, or N heteroatom. Preferably, heteroaryl is a 5- or 6-membered aromatic monocyclic ring in which one, two, or three carbon atoms are replaced with an O, N, or S heteroatom. Alternatively, heteroaryl refers to an aromatic bicyclic or tricyclic ring system in which one, two, three, four, or five carbon atoms are replaced with the same or different O, N, or S heteroatoms. Preferably, in each ring of a heteroaryl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, 1H-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, and the like. Includes zolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolidinyl, indolizinyl, indazolyl, purinyl, quinolidinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl and phenazinyl. Exemplary 5- or 6-membered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. "Substituted heteroaryl" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) hydrogen atoms of the heteroaryl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituent is a first-level substituent, a second-level substituent, or a third-level substituent as specified herein.

The term "heterocyclyl" or "heterocyclic ring" refers to a cycloalkyl group, as defined above, in which one, two, three, or four carbon atoms of the cycloalkyl group are replaced by a heteroatom of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. The heterocyclyl group preferably has one or two rings containing 3 to 10, e.g., 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of a heterocyclyl group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also intended to include partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the above heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also known as piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactone, lactam, cyclic imide, and cyclic anhydride. "Substituted heterocyclyl" means that one or more hydrogen atoms (e.g., from 1 to the maximum number of hydrogen atoms attached to the heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) of the heterocyclyl group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level, second-level, or third-level substituents as specified herein.

As used herein, the term "heterocycloalkylene" refers to a heterocyclyl group, as defined above, containing at least one ring heteroatom (e.g., selected from the group consisting of O, S, N, B, Si, and P) and in which one hydrogen atom has been removed to result in a geminal, vicinal, or isolated diradical. In certain embodiments, the heteroatom of a heterocycloalkylene group is selected from the group consisting of O, S, and N. For example, a heterocycloalkylene can be an O/S-heterocycloalkylene, e.g., an O-heterocycloalkylene. In certain embodiments, in each ring of a heterocycloalkylene group, the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. A heterocycloalkylene can be monocyclic or polycyclic (e.g., bi- or tricyclic). In certain embodiments, heterocycloalkylene is a mono-, bi-, or tricyclic 4- to 14-membered (i.e., 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered, e.g., 4- to 12- or 4- to 10-membered) heterocycloalkylene. The term "heterocycloalkylene" is also intended to encompass partially or fully hydrogenated forms (e.g., dihydro, tetrahydro, or perhydro forms) of the above heteroaryl groups in which one hydrogen atom is removed from the same carbon atom to provide a geminal diradical (preferably, partially or fully hydrogenated forms of the above mono-, bi-, or tricyclic heteroaryl groups). Thus, in certain embodiments, heterocycloalkylene is saturated or unsaturated (i.e., contains one or more double bonds of the heterocycloalkylene group within the ring), but cannot be aromatic. "Substituted heterocycloalkylene" means that one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the heterocycloalkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the non-hydrogen substituents are first-level substituents, second-level substituents, or third-level substituents specified herein.

As used herein, the phrase "partially hydrogenated form" of an unsaturated compound or group means that some unsaturation has been removed by formally adding hydrogen to an originally unsaturated compound or group without removing all of the unsaturation. The term "fully hydrogenated form" of an unsaturated compound or group is used interchangeably herein with the term "perhydro" and means that all unsaturation has been removed by formally adding hydrogen to an originally unsaturated compound or group. For example, a partially hydrogenated form of a 5-membered heteroaryl group (containing two double bonds in the ring, such as furan) includes a dihydro form of the 5-membered heteroaryl group (e.g., 2,3-dihydrofuran or 2,5-dihydrofuran), while a tetrahydro form of the 5-membered heteroaryl group (e.g., tetrahydrofuran, i.e., THF) is a fully hydrogenated (or perhydro) form of the 5-membered heteroaryl group. Similarly, for a 6-membered heteroaryl group having three double bonds in the ring (e.g., pyridyl), partially hydrogenated forms include di- and tetrahydro forms (e.g., di- and tetrahydropyridyl), while hexahydro forms (e.g., piperidinyl in the case of heteroarylpyridyl) are fully hydrogenated (or perhydro) derivatives of the 6-membered heteroaryl group. Consequently, hexahydro forms of an aryl or heteroaryl can only be considered partially hydrogenated forms according to the present invention if the aryl or heteroaryl contains at least four unsaturated moieties consisting of double and triple bonds between ring atoms.

The term "aromatic," as used in the context of hydrocarbons, requires that the entire molecule be aromatic. For example, if a monocyclic aryl is hydrogenated (partially or fully), the resulting hydrogenated ring structure is classified as a cycloalkyl for purposes of this invention. Similarly, if a bicyclic or polycyclic aryl (e.g., naphthyl) is hydrogenated, the resulting hydrogenated bicyclic or polycyclic structure (e.g., 1,2-dihydronaphthyl) is classified as a cycloalkyl for purposes of this invention (even though one ring, as in 1,2-dihydronaphthyl, is still aromatic). A similar distinction is made herein between heteroaryl and heterocyclyl. For example, indolinyl, i.e., the dihydro variant of indolyl, is classified as a heterocyclyl for purposes of this invention because only one ring of the bicyclic structure is aromatic and one of the ring atoms is a heteroatom.

The term "hydrocarbyl," as used herein, refers to a monovalent organic group obtained by removal of an H atom from a hydrocarbon molecule. In certain embodiments, the hydrocarbyl group is acyclic, e.g., linear (straight chain) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (e.g., arylalkyl (aralkyl)). Particular examples of hydrocarbyl groups are _C1-30 alkyl (e.g., _C6-30 alkyl, _C8-24 alkyl, or _C10-20 alkyl), _C2-30 alkenyl having one, two, or three double bonds (e.g., _C6-30 alkenyl, _C8-24 alkenyl, or _C10-20 alkenyl), aryl, and aryl( _C1-6 alkyl). In certain embodiments, the hydrocarbyl group is optionally substituted (e.g., with one or more first-level substituents, one or more second-level substituents, or one or more third-level substituents as defined herein), so long as the overall polarity of the hydrocarbon remains relatively non-polar. In certain embodiments, the hydrocarbyl group is the hydrocarbyl chain of a naturally occurring fatty acid and can have at least 8 carbon atoms, e.g., the hydrocarbyl group is a (preferably straight-chain) _C8-20 alkyl chain, e.g., a (preferably straight-chain) _C10-18 alkyl chain.

The term "optionally substituted" refers to a group in which one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or up to 10, e.g., 1 to 5, 1 to 4 or 1 to 3 or 1 or 2) hydrogen atoms are different from hydrogen (i.e., first level substituents), such as alkyl (preferably C _1-6 alkyl), alkenyl (preferably C _2-6 alkenyl), alkynyl (preferably C _2-6 alkynyl), aryl (preferably 6-14 membered aryl), heteroaryl (preferably 3-14 membered heteroaryl), cycloalkyl (preferably 3-14 membered cycloalkyl), heterocyclyl (preferably 3-14 membered heterocyclyl), halogen, -CN, azide, -NO ₂ , -OR ⁷¹ , -N(R ⁷² )(R ⁷³ ), -S(O) _0-2 R ⁷¹ , -S(O) _1-2 OR ⁷¹ , -OS(O) _1-2 R ⁷¹ , -OS(O) _1-2 OR ⁷¹ , -S(O) _1-2 N(R ⁷² )(R ⁷³ ), -OS(O) _1-2 N(R ⁷² )(R ⁷³ ), -N(R ⁷¹ )S(O) _1-2 R ⁷¹ , -NR ⁷¹ S(O) _1-2 OR ⁷¹ , -NR ⁷¹ S(O) _1-2 N(R ⁷² )(R ⁷³ ), -OP(O)(OR ⁷¹ ) ₂ , -C(=X ₁ )R ⁷¹ , -C(=X ₁ )X ₁ R ⁷¹ , —X ₁ C(═X ₁ )R ⁷¹ and —X ₁ C(═X ₁ )X ¹ R ⁷¹ and/or any two first-level substituents attached to the same carbon atom of a cycloalkyl or heterocyclyl group can together form ═X 1, wherein each of the alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl and heterocyclyl groups of the first-level substituents is itself selected from C _1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl, 3- to 14-membered cycloalkyl, 3- to 14-membered heterocyclyl, halogen, —CF 3 , —CN, azide, —NO 2 , —OR 81 , —N(R 82 )(R 83 ), —S(O) _0-2 R 81 , —S(O _{) 0-2} R 81 , —S(O _{) 0-2} R 82 , —S(O) 0-2 R 83 , —S(O) 0-2 R 84 , —S(O) 0-2 R 85 , —S(O) 0-2 R 86 , —S(O) 0-2 R 87 , —S(O) 0-2 R 88 , —S(O) 0-2 R ₈₉ , —S(O) 100 , —S(O) 110 , —S(O) 120 , —S(O) ¹³⁰ , —S(O ₎ ¹⁴⁰ , —S(O ⁾ 150 , —S(O) 160 , —S(O) 170 , —S(O) ₁₈₀ , —S(O) ¹⁹⁰ , —S _1-2 OR ⁸¹ , -OS(O) _1-2 R ⁸¹ , -OS(O) _1-2 OR ⁸¹ , -S(O) _1-2 N(R ⁸² )(R ⁸³ ), -OS(O) _1-2 N(R ⁸² )(R ⁸³ ), -N(R ⁸¹ )S(O) _1-2 R ⁸¹ , -NR ⁸¹ S(O) _1-2 OR ⁸¹ , -NR ⁸¹ S(O) _1-2 N(R ⁸² )(R ⁸³ ), -OP(O)(OR ⁸¹ ) ₂ , -C(=X ₂ )R ⁸¹ , -C(=X ₂ )X ₂ R ⁸¹ , -X ₂ C(=X ₂ )R and _/ or any two second-level substituents attached to the same carbon atom of a ^cycloalkyl or ^heterocyclyl group of the first-level substituents can together form =X ₂ , wherein each of the C 1-6 alkyl, C _2-6 alkenyl, C 2-6 alkynyl, 6- to 14-membered aryl, 3- to 14-membered heteroaryl _{, 3-} to 14-membered cycloalkyl, and 3- to 14-membered heterocyclyl groups of the second-level substituents optionally is C _1-3 alkyl, halogen, —CF ₃ , _—CN , azide, —NO ₂ , —OH, —O(C _1-3 alkyl), —OCF ₃ , —S(C _1-3 alkyl), —NH ₂ , —NH( _{C 1-3} alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —S(O) ₂ NH _2-z (C _1-3 alkyl) _z , —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)(C _1-3 alkyl), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _1-3 alkyl)C(═NH)NH _2-z (C _1-3 alkyl) _z (wherein each z is independently 0, 1 or 2, and each C _1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl), and/or any two third-level substituents attached to the same carbon atom of a 3- to 14-membered cycloalkyl or heterocyclyl group of the second-level substituent may together form ═O, ═S, ═NH, or ═N(C _1-3 alkyl);
where:
Each of R ⁷¹ , R ⁷² and R ⁷³ is independently selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3 to 7 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 7 membered heterocyclyl, wherein each of the C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 3 to 7 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 7 membered heterocyclyl groups optionally is C _1-3 alkyl, halogen, —CF ₃ , —CN, azide, —NO ₂ , —OH, —O(C _1-3 alkyl), —OCF ₃ , ═O, —S(C _1-3 alkyl), —NH ₂ , —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —S(O) ₂ NH _2-z (C _1-3 alkyl) _z , —C(═O)(C _1-3 alkyl), —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)( _{C 1-3 alkyl} ), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _1-3 alkyl)C(═NH)NH _2-z (C _1-3 alkyl) _z (wherein each z is independently 0, 1 or 2 and each C 1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl);
Each of R ⁸¹ , R ⁸² and R ⁸³ is independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 3 to 6 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 6 membered heterocyclyl, wherein each of the C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 3 to 6 membered cycloalkyl, 5 or 6 membered aryl, 5 or 6 membered heteroaryl and 3 to 6 membered heterocyclyl groups optionally is C _1-3 alkyl, halogen, —CF ₃ , —CN, azide, —NO ₂ , —OH, —O(C _1-3 alkyl), —OCF ₃ , ═O, —S(C _1-3 alkyl), —NH ₂ , —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —S(O) ₂ NH _2-z (C _1-3 alkyl) _z , —C(═O)(C _1-3 alkyl), —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)( _{C 1-3 alkyl} ), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _1-3 alkyl)C(═NH)NH _2-z (C _1-3 alkyl) _z (wherein each z is independently 0, 1 or 2 and each C 1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl); and X ₁ and X Each of ₂ is independently selected from O, S and N(R ⁸⁴ ), where R ⁸⁴ is H or C _1-3 alkyl.

Typical first level substituents are preferably C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, 6 to 14 membered (e.g. 6 to 10 membered) aryl, 3 to 14 membered (e.g. 5 or 6 membered) heteroaryl, 3 to 14 membered (e.g. 3 to 7 membered) cycloalkyl, 3 to 14 membered (e.g. 3 to 7 membered) heterocyclyl, halogen, —CN, azido, —NO ₂ , —OR ⁷¹ , —N(R ⁷² )(R ⁷³ ), —S(O) _0-2 R ⁷¹ , —S(O) _1-2 OR ⁷¹ , —OS(O) _1-2 R ⁷¹ , —OS(O) _1-2 OR ⁷¹ , —S(O) _1-2 N(R ⁷² )(R ⁷³ ), —OS(O) _1-2 N(R ⁷² )(R ⁷³ ), —N(R ⁷¹ )S(O) _1-2 R ⁷¹ , —NR ⁷¹ S(O) _1-2 OR ⁷¹ , —C(═X ₁ )R ⁷¹ , —C(═X ₁ )X ₁ R ⁷¹ , —X ₁ C(═X ₁ )R ⁷¹ and —X ₁ C(═X ₁ )X ₁ R ^{71 are} , for example, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 6-membered aryl, 5- or 6-membered heteroaryl, 3- to 7-membered cycloalkyl, 3- to 7-membered (for example 5- or 6-membered) heterocyclyl, halogen, —CF ₃ , —CN, azide, —NO ₂ , —OH, —O(C _1-3 alkyl), —S(C _1-3 alkyl), —NH ₂ , —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —S(O) ₂ NH _2-z (C _1-3 alkyl) _z , —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)(C _1-3 alkyl), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _1-3 alkyl)C(═NH)NH _2-z (C _1-3 alkyl) _z (wherein each z is independently 0, 1 or 2, and each C and X ₁ is independently selected from O, S, NH and N(CH ₃ ); and each of R ⁷¹ , R ⁷² and R ⁷³ is as defined above or preferably independently selected from the group consisting of H, C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 5- or 6-membered cycloalkyl, 5- or _6- membered aryl, 5- or 6-membered heteroaryl and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups optionally is C _1-3 alkyl, halogen, —CF ₃ , —CN, azide, —NO ₂ , —OH, —O(C _1-3 alkyl), —S(C _1-3 alkyl), —NH ₂ , —NH(C and -N(C _1-3 alkyl) _C ( ₌ NH)NH _{2- z (} C _1-3 alkyl) _{z ( wherein} each _{z is independently 0, 1 or 2 and each C 1-3 alkyl is independently methyl , ethyl , propyl or isopropyl} ) _. In certain embodiments, the first level substituents are selected from the group consisting of C _1-3 alkyl, phenyl, halogen, —CF ₃ , —OH, —OCH ₃ , —SCH ₃ , —NH _2-z (CH ₃ ) _z , —C(═O)OH and —C(═O)OCH ₃ (where z is 0, 1 or 2 and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl). In certain embodiments, the first level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl or Br) and —CF ₃ , for example halogen (e.g., F, Cl or Br) and —CF ₃ .

Typical second level substituents are preferably C _1-4 alkyl, C _2-4 alkenyl, C _2-4 alkynyl, 6- or 10-membered aryl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF ₃ , —CN, azido, —NO ₂ , —OH, —O(C _1-3 alkyl), —S(C _1-3 alkyl), —NH ₂ , —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —S(O) ₂ NH _2-z (C _1-3 alkyl) _z , —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)(C _1-3 alkyl), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _1-3 alkyl)C(═NH)NH _2-z (C _1-3 alkyl) _z (wherein each z is independently 0, 1 or 2, and each C _1-3 alkyl is independently methyl, ethyl, propyl, or isopropyl). Particular examples of second level substituents are C _1-3 alkyl, phenyl, 5- or 6-membered heteroaryl, 5- or 6-membered cycloalkyl, 5- or 6-membered heterocyclyl, halogen, ═O, ═S, —CF ₃ , —CN, —OH, —O(C _1-3 alkyl), —S(C _1-3 alkyl), —NH ₂ , —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , —NHS(O) ₂ (C _1-3 alkyl), —C(═O)OH, —C(═O)O(C _1-3 alkyl), —C(═O)NH _2-z (C _1-3 alkyl) _z , —NHC(═O)(C _1-3 alkyl), —NHC(═NH)NH _z-2 (C _1-3 alkyl) _z and —N(C _Particularly preferred _{second level} substituents are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl _, phenyl, ═O and ═S _.

Typical third-level substituents are preferably selected from the group consisting of C _1-3 alkyl, phenyl, halogen, —CF ₃ , —OH, —OCH ₃ , —SCH ₃ , —NH _2-z (CH ₃ ) _z , —C(═O)OH and —C(═O)OCH ₃ (where z is 0, 1 or 2 and C _1-3 alkyl is methyl, ethyl, propyl or isopropyl). Particularly preferred third-level substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl or Br) and —CF ₃ , for example halogen (e.g., F, Cl or Br) and —CF ₃ .

As used herein, the term "tertiary amine moiety" refers to a moiety containing a nitrogen atom substituted with three organic substituents, where the substituents may be the same or different from one another. In certain embodiments, the organic substituents are selected from hydrocarbyl groups (e.g., alkyl groups, particularly C 1-6 alkyl groups) that are optionally substituted (e.g., with one or more first-level substituents, one or more second-level substituents, or one or more _third -level substituents as defined herein).

As used herein, the term "filtration" refers to any process involving the removal or separation of at least one component (e.g., permeable molecules such as salts, small proteins, solvents, etc.) of a liquid composition based on the molecular size of the component contained in the composition. This separation may use small molecule-permeable filters (e.g., for diafiltration or tangential flow filtration) or semipermeable membranes (e.g., dialysis). Thus, examples of filtration include dialysis, tangential flow filtration, and diafiltration.

As used herein, the phrase "substantially free of X" means that the mixture (e.g., a composition described herein or its aqueous phase) is substantially and practically free of X. For example, if the mixture is substantially free of X, the amount of X in the mixture can be less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the mixture.

For example, as used herein, "substantially free of lipids comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeat units" means that the mixture (e.g., a composition described herein or its aqueous phase) is virtually and practically free of lipids comprising at least 30 consecutive ethylene glycol repeat units in a manner that is practically feasible. For example, if the mixture is substantially free of lipids comprising at least 30 consecutive ethylene glycol repeat units, the amount of lipids comprising at least 30 consecutive ethylene glycol repeat units in the mixture can be less than 1 wt. % (e.g., less than 0.5 wt. %, less than 0.4 wt. %, less than 0.3 wt. %, less than 0.2 wt. %, less than 0.1 wt. %, less than 0.09 wt. %, less than 0.08 wt. %, less than 0.07 wt. %, less than 0.06 wt. %, less than 0.05 wt. %, less than 0.04 wt. %, less than 0.03 wt. %, less than 0.02 wt. %, less than 0.01 wt. %, less than 0.005 wt. %, or less than 0.001 wt. %) based on the total weight of the mixture. Similar considerations apply to phrases that include the term "substantially free" (e.g., "substantially free of sarcosylated lipids," "substantially free of (POX)-conjugated and/or polyoxazine (POZ)-conjugated lipids," and "substantially free of any polymer-conjugated lipids other than amphiphilic OEG-conjugated compounds").

The phrase "nucleic acid integrity" refers to the percentage of full-length (i.e., unfragmented) nucleic acids relative to the total amount of nucleic acid (i.e., unfragmented + fragmented nucleic acids) contained in a sample. Nucleic acid integrity can be determined by chromatographically separating the nucleic acids (e.g., using capillary electrophoresis), determining the peak area of the main nucleic acid peak (i.e., the peak area of the full-length (i.e., unfragmented) nucleic acid), determining the peak area of the total nucleic acid, and dividing the peak area of the main nucleic acid peak by the peak area of the total nucleic acid. Similarly, the phrase "RNA integrity" refers to the percentage of full-length (i.e., unfragmented) RNA relative to the total amount of RNA (i.e., unfragmented + fragmented RNA) contained in a sample. RNA integrity can be determined by chromatographically separating the RNA (e.g., using capillary electrophoresis), determining the peak area of the major RNA peak (i.e., the peak area of full-length (i.e., unfragmented) RNA), determining the peak area of total RNA, and dividing the peak area of the major RNA peak by the peak area of total RNA.

The term "cryoprotectant" refers to a substance added to a formulation (e.g., a preparation or composition) to protect the active ingredients of the formulation during the freezing step.

The term "lyoprotectant" refers to a substance added to a formulation to protect the active ingredient during the drying stage.

According to the present invention, the term "peptide" includes oligo- and polypeptide-containing substances comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The terms "protein" or "polypeptide" include large peptides, particularly peptides having at least about 151 amino acids, and the terms "peptide," "polypeptide," and "protein" are generally used synonymously herein.

A "therapeutic peptide or protein," when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the subject's condition or disease state. In certain embodiments, a therapeutic peptide or protein has curative or palliative properties and can be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "therapeutic peptide or protein" includes whole peptides or proteins, as well as therapeutically active fragments thereof. It can also include therapeutically active variants of peptides or proteins. Examples of therapeutically active peptides or proteins include, but are not limited to, vaccination antigens and immune stimulants such as cytokines. The terms "therapeutic peptide or protein" and "pharmaceutically active peptide or protein" are used interchangeably herein.

The term "portion" refers to a fraction. With respect to a particular structure, such as an amino acid sequence or a protein, the term "portion" may designate a continuous or discontinuous fraction of that structure.

The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure such as an amino acid sequence or protein refers to a continuous element of that structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

A "fragment" of an amino acid sequence (peptide, polypeptide, or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing the amino acid sequence truncated at the N-terminus and/or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon for initiating translation. A fragment of an amino acid sequence contains, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, a sequence of up to 8, particularly up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

According to the present invention, a portion or fragment of a peptide, polypeptide, or protein preferably possesses at least one functional property of the peptide, polypeptide, or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides, polypeptides, or proteins, enzymatic activity, interaction with antibodies, and selective binding of nucleic acids. For example, a pharmacologically active fragment of a peptide, polypeptide, or protein possesses at least one pharmacological activity of the peptide, polypeptide, or protein from which it is derived. A portion or fragment of a peptide, polypeptide, or protein preferably comprises a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids of the peptide or protein. A portion or fragment of a peptide or protein preferably comprises a sequence of at most 8, particularly at most 10, at most 12, at most 15, at most 20, at most 30, or at most 55 consecutive amino acids of the peptide or protein.

As used herein, a "variant" with respect to an amino acid sequence (peptide, polypeptide, or protein) refers to an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid or a modification of the same amino acid). The parent amino acid sequence may be a naturally occurring or wild-type (WT) amino acid sequence or may be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences, and preferably 1 to about 10 or 1 to about 5 amino acid differences compared to the parent.

"Wild-type" or "WT" or "native" with respect to an amino acid sequence means an amino acid sequence found in nature, including allelic variations. A wild-type amino acid sequence, peptide, polypeptide, or protein has an amino acid sequence that has not been intentionally modified. Similarly, "wild-type" or "WT" or "native" with respect to a nucleic acid sequence means a nucleic acid sequence that has been found in nature, including allelic variations. For example, a wild-type coding sequence means a coding sequence that is found in nature and has not been intentionally modified.

As used herein, "coding sequence" means a portion of a nucleic acid (e.g., the DNA or RNA of a gene) that encodes a protein.

The phrase "guanosine/cytosine (G/C) content" or "G/C content" refers to the percentage of bases in a DNA or RNA molecule that are guanine (G) or cytosine (C). The G/C content can be given for a specific portion of DNA or RNA or for the entire genome. When referring to a portion, the G/C content can refer to the G/C content of an individual gene or portion (domain) of a gene, a group or cluster of genes, a non-coding region, a coding sequence, or a synthetic oligonucleotide such as a primer.

For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. The term "variant" includes total mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those occurring naturally. The term "variant" particularly includes fragments of an amino acid sequence.

Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion and appropriate screening of the resulting products are also possible. Amino acid addition variants include amino- and/or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and/or C-terminal ends of the protein are also referred to herein as N- and/or C-terminal truncated variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. It is preferred to modify and/or replace amino acids with others having similar properties at positions in the amino acid sequence that are not conserved between homologous proteins or peptides. In some embodiments, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of one amino acid within a family of amino acids with related side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups:
- glycine, alanine;
- valine, isoleucine, leucine;
- aspartic acid, glutamic acid;
- asparagine, glutamine;
- serine, threonine;
- lysine, arginine; and - phenylalanine, tyrosine.

In one embodiment, the degree of similarity, preferably identity, between an amino acid sequence and an amino acid sequence that is a variant of the amino acid sequence is at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably expressed over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably expressed for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, in some embodiments, consecutive amino acids. In some embodiments, the degree of similarity or identity is expressed for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using best sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, and Gap Extend 0.5.

"Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

The terms "% identical" and "% identity" or similar terms refer specifically to the percentage of nucleotides or amino acids that are identical in optimal alignment between the compared sequences. The percentage is purely statistical; differences between the two sequences may, but are not necessarily, randomly distributed throughout the length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequence. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.). In some embodiments, the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithm available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq). In some embodiments, the algorithm parameters used with the BLASTN algorithm on the NCBI website include: (i) a prediction threshold setting of 10; (ii) a word length setting of 28; (iii) a maximum match of query scope setting of 0; (iv) a match/mismatch score setting of 1, -2; (v) a gap cost setting of linear; and (vi) use of a filter for low complexity regions. In one embodiment, the algorithm parameters used with the BLASTP algorithm on the NCBI website include: (i) a prediction threshold setting of 10; (ii) a word length setting of 3; (iii) a maximum query range match setting of 0; (iv) a matrix setting of BLOSUM62; (v) gap cost settings of presence: 11, expansion: 1; and (vi) a conditional composition score matrix adjustment.

Percentage identity is obtained by determining the number of identical positions in the compared sequences, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

In some embodiments, the degree of similarity or identity is expressed over a region of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is expressed over at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 nucleotides, in some embodiments, consecutive nucleotides. In some embodiments, the degree of similarity or identity is expressed over the entire length of the reference sequence.

According to the present invention, a homologous amino acid sequence exhibits at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

The amino acid sequence variants described herein can be readily produced by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily produced with the aid of known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

In certain embodiments, a fragment or variant of an amino acid sequence (peptide, polypeptide, or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., functionally equivalent. With respect to antigens or antigenic sequences, a particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers to a variant molecule or sequence that contains an amino acid sequence in which one or more amino acids have been altered, particularly compared to the amino acid sequence of the parent molecule or sequence, yet still retains one or more functions of the parent molecule or sequence, e.g., induction of an immune response (an immunogenic fragment). In certain embodiments, modifications to the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present; for example, the immunogenicity of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence. However, in other embodiments, the immunogenicity of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. In certain embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, one of skill in the art will understand that antigens suitable for use herein can be altered from the naturally occurring or native sequence from which they are derived while maintaining the desired activity of the native sequence.

In certain embodiments, "isolated" refers to alteration or removal (e.g., purification) from a natural state or an artificial composition, such as a composition from a manufacturing process. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as, for example, a host cell. In certain embodiments, RNA (e.g., mRNA) used in the present invention is in a substantially purified form. In certain embodiments, a solution (preferably an aqueous solution) of RNA (e.g., mRNA) in a substantially purified form comprises a first buffer system.

The term "genetic modification" or simply "modification" includes the transfection of a cell with a nucleic acid.

The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" may also include the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, where the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection of nucleic acids described herein can be present in vitro (e.g., cell culture) or in vivo; for example, the cells may form part of a patient's organ, tissue, and/or organism. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient if the transfected genetic material is expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during transfection are not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded via mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection should be performed. Such stable transfection can be achieved using viral or transposon-based systems for transfection. Generally, antigen-encoding nucleic acids are transiently transfected into cells. RNA can be transfected into cells to transiently express its encoded protein.

The present invention includes analogs of peptides, polypeptides, or proteins. According to the present invention, a peptide, polypeptide, or protein analog is a modified form of the peptide, polypeptide, or protein derived from the peptide, polypeptide, or protein and retains at least one functional property. For example, a pharmacologically active peptide, polypeptide, or protein analog retains at least one pharmacological activity of the peptide, polypeptide, or protein from which the analog is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and/or additions of any molecule associated with the protein, polypeptide, or peptide, such as carbohydrates, lipids, and/or proteins or peptides. In certain embodiments, a "protein, polypeptide, or peptide" "analog" includes modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting/blocking groups, proteolytic cleavage, or binding to antibodies or other cellular ligands. The term "analog" also extends to fully functional chemical equivalents of the proteins, polypeptides, and peptides.

As used herein, the terms "bonded," "fused," and "fused" are used interchangeably. These terms refer to the joining of two or more elements, components, or domains.

According to various embodiments of the present invention, a nucleic acid, such as RNA (e.g., mRNA), encoding a peptide, polypeptide, or protein is taken up or introduced, i.e., transfected or transduced, into a cell, which may be in vitro or present in a subject, resulting in expression of the peptide, polypeptide, or protein. The cell may express the encoded peptide, polypeptide, or protein intracellularly (e.g., in the cytoplasm and/or nucleus) and may secrete and/or express the encoded peptide, polypeptide, or protein on its surface.

According to the present invention, terms such as "expressing nucleic acid" and "encoding nucleic acid" or similar terms are used interchangeably herein and mean that, with respect to a particular peptide, polypeptide, or protein, the nucleic acid can be expressed to produce the peptide, polypeptide, or protein when present in an appropriate environment, preferably within a cell.

As used herein, "activation" or "stimulation" refers to the state of a cell (e.g., an immune effector cell, such as a T cell) that has been stimulated sufficiently to induce detectable cell proliferation. Activation can also be associated with the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cell" refers, inter alia, to an immune effector cell that is undergoing cell division.

The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, triggering its differentiation into an effector cell, such as an effector T cell.

The terms "clonal expansion" or "expansion" refer to the process of multiplication of a specific entity. In one embodiment, the term is preferably used in the context of an immunological response, in which immune effector cells are stimulated by an antigen, proliferate, and the specific immune effector cells that recognize the antigen are expanded. In one embodiment, expansion leads to differentiation of the immune effector cells.

According to the present invention, "antigen" refers to any substance that elicits an immune response and/or any substance to which an immune response or mechanism is directed, such as a cellular response and/or a humoral response. This also includes situations in which an immune response or mechanism is directed against one or more antigenic peptides, particularly if the antigen is processed into antigenic peptides and presented in the context of MHC molecules. In particular, "antigen" relates to any substance, preferably a peptide or protein, that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" can include any molecule that contains at least one epitope, such as a T cell epitope. In certain embodiments, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In certain embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

According to the present invention, any suitable antigen that is a candidate for an immune response can be used, where the immune response can be a humoral or cellular immune response, or both. In the context of certain embodiments of the present invention, the antigen is presented by a cell, preferably an antigen-presenting cell, in the context of an MHC molecule, and an immune response against the antigen is generated. The antigen can correspond to or be a product derived from a naturally occurring antigen. Such naturally occurring antigens can include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen can also be a tumor antigen. According to the present invention, the antigen can correspond to a naturally occurring product, for example, a viral protein or a portion thereof.

The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (e.g., bacterial or viral antigens), or antigens associated with cancer, such as tumor antigens, typically tumors.

In one embodiment, the antigen is a tumor antigen, i.e., one that occurs as part of a tumor cell, particularly primarily as an intracellular or surface antigen of the tumor cell. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., a virus, bacterium, unicellular organism, or parasite, e.g., a viral antigen such as a viral ribonucleoprotein or coat protein. In particular, the antigen should be presented by an MHC molecule, which leads to the modulation, particularly activation, of cells of the immune system, preferably CD4+ and CD8+ lymphocytes, particularly via modulation of the activity of T-cell receptors.

The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to an antigen produced as an intracellular or surface antigen in a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin and fetal sulfoglycoprotein, α2-H-ferroprotein and γ-fetoprotein, as well as various viral tumor antigens. According to one embodiment of the present invention, tumor antigens include any antigen that is characteristic of a tumor or cancer and that is characteristic of tumor or cancer cells in terms of type and/or expression level.

The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of inducing an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

The term "epitope" refers to an antigenic determinant on a molecule such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can comprise a continuous or discontinuous portion of the protein and can be, for example, about 5 to about 100, about 5 to about 50, about 8 to about 0, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, an epitope in the context of the present invention is a T-cell epitope.

Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and refer to, for example, an incomplete presentation of an antigen, e.g., that elicits an immune response to the antigen or that enables a cell to express, contain, and present the antigen. In certain embodiments, the term refers to an immunogenic portion of an antigen. Preferably, this is the portion of the antigen that is recognized (i.e., specifically binds) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 8 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized when presented in the context of a T cell's MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells in an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and present both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC/peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. For class II MHC/peptide complexes, binding peptides are typically about 10 to about 25 amino acids in length, and particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may also be effective.

Peptide and protein antigens can be, for example, 2 to 100 amino acids in length, including 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

A peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to generate antibodies and T cell responses against the peptide or protein.

In certain embodiments, the vaccine antigen, i.e., the antigen whose inoculation into a subject induces an immune response, is recognized by immune effector cells. In certain embodiments, the vaccine antigen, if recognized by immune effector cells, can, in the presence of appropriate costimulatory signals, induce the stimulation, priming, and/or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of this embodiment of the invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably an antigen-presenting cell.

In some embodiments, the antigen is expressed on diseased cells (e.g., tumor cells or infected cells).

In some embodiments, the antigen is presented by a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of MHC. In some embodiments, when expressed by and/or presented to a T cell, binding of the TCR to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and/or expansion of the T cell. In some embodiments, when expressed by and/or presented to a T cell, binding of the TCR to an antigen presented by a diseased cell results in cytolysis and/or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, e.g., perforin and granzymes.

In some embodiments, the antigen is expressed on the surface of a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope in the extracellular domain. In some embodiments, the CAR binds to an epitope of a natural antigen present on the surface of a living cell. In some embodiments, when expressed by and/or presented to a T cell, binding of the CAR to an antigen presented on a cell, such as an antigen-presenting cell, results in stimulation, priming, and/or expansion of the T cell. In some embodiments, when expressed by and/or presented to a T cell, binding of the CAR to an antigen presented on a diseased cell results in cytolysis and/or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, e.g., perforin and granzymes.

In some embodiments, the antigen receptor is an antibody or B cell receptor that binds to an epitope on an antigen. In some embodiments, the antibody or B cell receptor binds to an epitope on a native antigen.

The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as an antigen, is associated with and located on the plasma membrane of a cell, where at least a portion of the molecule faces the extracellular space of the cell and is accessible from outside the cell, e.g., by an antibody located outside the cell. In this context, a portion can be, for example, at least 4, at least 8, at least 12, or at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interactions with any other proteins, lipids, saccharides, or other structures that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule that associates with the cell surface can be a transmembrane protein having an extracellular portion, or a protein that associates with the cell surface through interactions with another protein that is a transmembrane protein.

"Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art, and thus includes the outside of a cell that is accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and is accessible for binding, for example, by an antigen-specific antibody added to the cell. In some embodiments, an antigen expressed on the cell surface is an integral membrane protein having an extracellular portion that can be recognized by a CAR.

The term "extracellular portion" or "extracellular domain" in the context of the present invention refers to a portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, for example, by a binding molecule, such as an antibody, that is located on the outside of the cell. In certain embodiments, the term refers to one or more extracellular loops or domains or fragments thereof.

The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes its target antigen, particularly when presented on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, in the context of an MHC molecule, and preferably exerts a T cell effector function. A T cell is considered specific for an antigen if the cell kills a target cell that expresses the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured. In certain embodiments of the invention, the RNA (particularly mRNA) encodes at least one epitope.

The term "target" refers to a factor, such as a cell or tissue, that is the target of an immune response, such as a cellular immune response. Targets include cells that present an antigen or an antigen epitope, i.e., a peptide fragment derived from an antigen. In one embodiment, the target cell is a cell that expresses an antigen, preferably presenting the antigen in conjunction with class I MHC.

"Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides) and the association (e.g., via binding) of these fragments with MHC molecules for presentation to specific T cells by one or more cells, preferably antigen-presenting cells. Antigen-presenting cells can be divided into professional and non-professional antigen-presenting cells.

The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon-gamma. Examples of non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample their environment for pathogens, such as viruses and bacteria. Upon contact with a presentable antigen, they are activated into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on the cell surface using MHC molecules. Concurrently, they upregulate cell surface receptors that act as coreceptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces migration of dendritic cells to the spleen via the bloodstream or to lymph nodes via the lymphatic system. Here, they act as antigen-presenting cells by presenting antigens, and activate helper T cells, killer T cells, and B cells in conjunction with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T cell- or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically phagocytose and kill foreign pathogens within the macrophage through hydrolytic and obstetric attack, resulting in their degradation. Peptides from degraded proteins are presented on the macrophage cell surface where they can be recognized by T cells and can directly interact with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

"Antigen-responsive CTL" means a CD8 ⁺ T cell that responds to an antigen or a peptide derived from said antigen that is presented together with class I MHC on the surface of an antigen-presenting cell.

According to the present invention, CTL responsiveness can induce sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic cell death of tumor antigen-expressing target cells. CTL responsiveness can also be determined using artificial reporters that accurately represent CTL responsiveness.

The terms "immune response" and "immune reaction" are used interchangeably herein in their conventional sense and refer to the body's integrated response to an antigen, and may refer to a cellular immune response, a humoral immune response, or both. According to the present invention, the terms "immune response to" or "immune response against," in reference to an agent such as an antigen, cell, or tissue, refer to an immune response, such as a cellular response, directed toward the agent. An immune response may include one or more responses selected from the group consisting of the development of antibodies against one or more antigens and the expansion of antigen-specific T lymphocytes, such as CD4 ^{+ and CD8+ T} lymphocytes, e.g., CD8+ T lymphocytes, which may be detected in vitro by various proliferation or cytokine production tests.

The terms "induction of an immune response" and "elicitation of an immune response" and similar terms in the context of the present invention refer to the induction of an immune response, e.g., the induction of a cellular immune response, a humoral immune response, or both. The immune response can be protective/preventative/prophylactic and/or therapeutic. The immune response can be directed against any immunogen or antigen or antigenic peptide, preferably a tumor-associated antigen or a pathogen-associated antigen (e.g., an antigen of a virus (e.g., influenza virus (A, B, or C), CMV, or RSV)). "Induction" in this context can mean that there was no immune response against a particular antigen or pathogen before induction, but it can also mean that there was a level of immune response against a particular antigen or pathogen before induction, and that the immune response is enhanced after induction. Thus, "induction of an immune response" in this context includes "enhancing an immune response." In some embodiments, after induction of an immune response in an individual, the individual is protected from developing a disease, such as an infectious disease or a cancerous disease, or the disease state is alleviated due to the induction of an immune response.

The terms "cellular immune response,""cellularresponse,""cell-mediatedimmunity," or similar terms are intended to include cellular responses directed against cells characterized by expression of antigens and/or presentation of antigens with class I or class II MHC. Cellular responses involve cells called T cells or T lymphocytes that act as "helpers" or "killers." Helper T cells (also called CD4 ⁺ T cells) play a central role in regulating the immune response, while killer cells (also called cytotoxic T cells, cytolytic T cells, CD8 ⁺ T cells, or CTLs) kill cells, such as diseased cells.

The term "humoral immune response" refers to the process in living organisms by which antibodies are generated in response to factors and organisms that are ultimately neutralized and/or eliminated. The specificity of the antibody response is mediated by T and/or B cells through membrane-associated receptors that bind to a single specific antigen. After binding the appropriate antigen and receiving various other activation signals, B lymphocytes divide and produce memory B cells as well as antibody-secreting plasma cell clones, each of which produces antibodies that recognize the same antigenic epitope recognized by its antigen receptor. Memory B lymphocytes remain quiescent until subsequently activated by a specific antigen. These lymphocytes provide the cellular basis of memory and result in an increased antibody response upon re-exposure to the specific antigen.

As used herein, the term "antibody" refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, capable of specifically binding to an epitope of an antigen under typical physiological conditions, preferably with a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days or more, etc., or any other relevant, functionally defined period of time (e.g., a period of time sufficient for the antibody to induce, promote, enhance, and/or modulate an antigen-associated physiological response to the antigen and/or a period of time sufficient for the antibody to recruit effector activity). In particular, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, chimeric antibodies, and any combination thereof. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The variable and constant regions are also referred to as variable and constant domains, respectively. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs of the VH are referred to as HCDR1, HCDR2, and HCDR3, and the CDRs of the VL are referred to as LCDR1, LCDR2, and LCDR3. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of an antibody comprises a heavy chain constant region (CH) and a light chain constant region (CL), where CH can be further subdivided into a constant domain CH1, a hinge region, and constant domains CH2 and CH3 (arranged in the following order from amino terminus to carboxy terminus: CH1, CH2, CH3). The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or may be an immunologically active portion of an intact immunoglobulin. An antibody is typically a tetramer of immunoglobulin molecules. Antibodies may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab) ₂ , as well as single-chain antibodies and humanized antibodies.

The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domains that interact with an antigen. The terms "binding region" and "antigen-binding region" are used interchangeably herein and refer to the region that interacts with an antigen and includes both the VH and VL regions. As used herein, antibodies include not only monospecific antibodies, but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions.

As noted above, the term antibody herein, unless otherwise specified or clearly contradicted by context, includes antigen-binding fragments, i.e., fragments of antibodies that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term "antibody" are: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains or the monovalent antibodies disclosed in WO 2007/059782 (Genmab); (ii) F(ab') ₂ fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting essentially of the VH and CH1 domains; (iv) Fv fragments consisting essentially of the VL and VH domains of a single-arm antibody; (v) dAb fragments consisting essentially of the VH domain (Ward et al., Nature 341 , 544-546 (1989)) and also known as domain antibodies (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90); (vi) camelid or nanobody molecules (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24); and (vii) isolated complementarity-determining regions (CDRs). Furthermore, the two domains of an Fv fragment, VL and VH, are encoded by separate genes but can be linked using recombinant methods by a synthetic linker that allows the VL and VH regions to be produced as a single protein chain that pairs to form a monovalent molecule (known as single-chain antibodies or single-chain Fvs (scFvs), see, e.g., Bird et al., Science 242 , 423-426 (1988) and Huston et al., PNAS USA 85 , 5879-5883 (1988)). Such single-chain antibodies are included within the scope of the term antibody unless otherwise specified or clearly contradicted by the context. While such fragments are generally included within the meaning of antibody, collectively and each independently are unique features of the present invention and exhibit various biological properties and utilities. These and other useful antibody fragments and bispecific forms of such fragments in the context of the present invention are further described herein.
It should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to antigens, which may be provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques.

The term "immunoglobulin" relates to proteins of the immunoglobulin superfamily, e.g., antibodies or antigen receptors such as B-cell receptors (BCRs). Immunoglobulins are characterized by structural domains, i.e., immunoglobulin domains, with a characteristic immunoglobulin (Ig) fold. The term includes membrane-bound immunoglobulins as well as soluble immunoglobulins. Membrane-bound immunoglobulins are also called surface or membrane immunoglobulins and are generally part of the BCR. Soluble immunoglobulins are generally referred to as antibodies. Immunoglobulins generally comprise several chains, typically two identical heavy chains and two identical light chains linked by disulfide bonds. These chains consist mainly of immunoglobulin domains such as _VL (variable light chain), _CL (constant light chain), _VH (variable heavy chain), and _CH (constant heavy chain) domains _CH1 , _CH2 , _CH3 , and _CH4 . There are five types of mammalian immunoglobulin heavy chains, namely α, δ, ε, γ, and μ, which constitute the different classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM. In contrast to the heavy chains of soluble immunoglobulins, the heavy chains of membrane or surface immunoglobulins contain a transmembrane domain and a short cytoplasmic domain at the carboxy terminus. In mammals, there are two types of light chains, namely lambda and kappa. Immunoglobulin chains contain a variable region and a constant region. The constant region is essentially conserved within the various isotypes of immunoglobulins, while the variable portion is highly diverse and is responsible for antigen recognition.

The terms "vaccination" and "immunization" refer to the process of treating an individual for therapeutic or prophylactic reasons, and relate to a method of administering to an individual one or more immunogens or antigens described herein or derivatives thereof, particularly in the form of RNA (especially mRNA) encoding same, to stimulate an immune response against said one or more immunogens or antigens or cells characterized by presentation of said one or more immunogens or antigens.

"Cells characterized by antigen presentation" or "cells presenting antigen" or "MHC molecules presenting antigens on the surface of antigen-presenting cells" or similar expressions mean that cells or antigen-presenting cells, such as diseased cells, particularly tumor or infected cells, present antigens or antigenic peptides, either directly or after processing, in the context of MHC molecules, preferably MHC class I and/or MHC class II molecules, most preferably MHC class I molecules.

In the present invention, the term "transcription" refers to the process by which the genetic code of a DNA sequence is transcribed into RNA (especially mRNA). RNA (especially mRNA) can then be translated into peptides, polypeptides or proteins.

As used herein, the term "expression" is defined as the transcription and/or translation of a specific nucleotide sequence.

With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes in which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

In the present invention, the term "RNA encodes" means that the RNA, when present in an appropriate environment, such as within the cells of a target tissue, is capable of directing the assembly of amino acids to produce the peptide or protein it encodes during the process of translation.

As used herein, the term "serum" refers to a fluid resulting from the removal of cells and clotting factors from whole blood, such as whole blood obtained from a human or mouse. In some embodiments, the serum is human serum or mouse serum.

The pharmaceutical preparations, particularly kits, described herein may include instructional materials or directions. As used herein, "instruction materials" or "instructions" includes publications, records, diagrams, or any other means of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional materials of the kits of the present invention may, for example, be attached to a container containing a composition of the present invention or shipped together with a container containing the composition. Alternatively, the instructional materials may be shipped separately from the container, with the intention that the instructional materials and the composition be used cooperatively by the recipient.

As used herein, the term "optionally" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and the description includes instances where the event, circumstance, or condition occurs and instances where it does not occur.

A prodrug of a particular compound described herein is a compound that undergoes chemical conversion under physiological conditions to provide the particular compound after administration to an individual. Additionally, a prodrug can be converted to the particular compound by chemical or biochemical methods in an ex vivo environment. For example, a prodrug can be slowly converted to the particular compound when placed in a transdermal patch reservoir with, for example, a suitable enzyme or chemical reagent. Exemplary prodrugs are in vivo hydrolyzable esters (using an alcohol or carboxy group contained in the particular compound) or amides (using an amino or carboxy group contained in the particular compound). Specifically, certain amino groups contained in the particular compound that bear at least one hydrogen atom can be converted to a prodrug form. Exemplary N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

As used herein, the structural formula of a compound may represent a certain isomer of the compound. However, it should be understood that the present invention includes all isomers and mixtures of isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and other structurally occurring isomers, and is not limited to the description of the formula. Furthermore, as used herein, the structural formula of a compound may represent a specific salt and/or solvate of the compound. However, it should be understood that the present invention includes all salts (e.g., pharmaceutically acceptable salts) and solvates (e.g., hydrates), and is not limited to the description of a specific salt and/or solvate.

"Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or the geometric (spatial) arrangement of functional groups and/or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains a pair of equal amounts of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert into each other by the migration of individual atoms or groups of atoms; i.e., tautomers are in dynamic chemical equilibrium with each other. Examples of tautomers are keto-enol-tautomeric isomers. "Conformers" are stereoisomers that can be interconverted only by rotation about a single bond, leading to—in particular—different three-dimensional configurations of (hetero)cyclic rings, such as chair, half-chair, boat, and twist-boat forms of cyclohexane.

As used herein, the term "solvate" refers to an addition complex of a dissolved substance in a solvent (e.g., an organic solvent (e.g., an aliphatic alcohol (e.g., methanol, ethanol, n-propanol, isopropanol), acetone, acetonitrile, ether, etc.), water, or a mixture of two or more of these liquids), where the addition complex exists in crystalline or mixed crystalline form. The amount of solvent present in the addition complex can be stoichiometric or non-stoichiometric. A "hydrate" is a solvate in which the solvent is water.

In an isotopically labeled compound, one or more atoms have the same number of protons but are replaced with corresponding atoms with different neutrons. For example, a hydrogen atom can be replaced with a deuterium or tritium atom. Exemplary isotopes that can be used in the present invention include deuterium, tritium, ^11C , ^13C , ^14C , ^15N , ^18F , ^32P , ^32S , ^35S , ^36Cl and ^125I .

The term "average diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) and data analysis using the so-called cumulant algorithm, which results in the so-called Z- _average and length dimensions and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Herein, the "average diameter,""diameter," or "size" of a particle is used synonymously with this value of Z- _average .

In one embodiment, the "polydispersity index" is calculated based on dynamic light scattering measurements by the so-called cumulant analysis described in the definition of "average diameter." Under certain conditions, it can be interpreted as a measure of the size distribution of the entire nanoparticles.

The "cross-sectional radius of gyration" of a particle about an axis of rotation (abbreviated here as _Rg ) is the radial distance from the point on that axis of rotation at which the moment of inertia about that axis is assumed to be the same as the actual mass distribution if the entire mass of the particle were assumed to be concentrated. Mathematically, _Rg is the root-mean-square distance of the particle's components from the center of mass or an axis. For example, for a macromolecule consisting of n mass elements of mass _m, where i = 1, 2, 3, ..., n, located at a fixed distance _s from the center of mass, _Rg is the square root of the mass average ^of _s over all mass elements and can be calculated as follows:

The cross-sectional radius of gyration can be empirically determined or calculated, for example, using light scattering. In particular, the small scattering vector
For , the structure function S is determined as follows:
(where N is the number of components (Guinier's rule).)

The "D10 value" is the diameter below which 10% of the particles have a diameter, particularly in relation to the quantitative size distribution of particles. The D10 value is meant to refer to the proportion of the smallest particles within a particle population (e.g., within a particle peak obtained by a flow field separation method).

The "D50 value" is the diameter below which 50% of the particles have a diameter, particularly in relation to a quantitative particle size distribution. The D50 value is meant to refer to the average particle size of a particle population (e.g., within a particle peak obtained by a flow field separation method).

The "D90 value" is the diameter below which 90% of the particles have a diameter, particularly in relation to the quantitative size distribution of particles. The "D95", "D99" and "D100" values have corresponding meanings. The D90, D95, D99 and D100 values are meant to refer to the proportion of large particles within a particle population (e.g., within a particle peak obtained by a field-flow separation method).

The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a small, highly hydrated charged particle may have a larger hydrodynamic radius than a large, weakly hydrated charged particle. This is because the small particle will entrain a greater number of water molecules as it moves through the solution. Because the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius is determined using the Stokes-Einstein equation:
where _kB is the Boltzmann constant; T is the temperature; η is the viscosity of the solvent; and D is the diffusion coefficient.
The diffusion coefficient can be determined empirically, for example, by dynamic light scattering (DLS). Thus, one method for determining the hydrodynamic radius of a particle or particle population (e.g., the hydrodynamic radius of a particle such as an LNP contained in a formulation or composition described herein, or the hydrodynamic radius of a particle peak obtained by subjecting such a formulation or composition to a flow-field separation method) is to measure the DLS signal of the particle or particle population (e.g., the DLS signal of a particle such as an LNP contained in a formulation or composition described herein, or the DLS signal of a particle peak obtained by subjecting such a formulation or composition to a flow-field separation method).

As used herein, the term "aggregate" refers to a mass of particles in which the particles are identical or very similar and are adhered to each other in a non-covalent manner (e.g., ionic interactions, H-bridge interactions, dipole interactions, and/or van der Waals interactions).

As used herein, the expression "light scattering" refers to the physical process by which light is deviated from its linear trajectory by one or more paths due to localized non-uniformities in the medium through which it passes.

The term "UV" means ultraviolet light, a band of the electromagnetic spectrum having wavelengths between 10 nm and 400 nm, i.e., shorter than visible light but longer than X-rays.

The expression "multi-angle light scattering" or "MALS" as used herein refers to a technique for measuring by scattering light from a sample at multiple angles. "Multi-angle" in this context means that scattered light can be detected at different discrete angles, measured, for example, by a single detector moving over a range including a selected specific angle, or by a series of detectors fixed at a specific angular position and including the specific angle. In a preferred embodiment, the light source used in MALS is a laser source (MALLS: multi-angle laser light scattering). Based on the MALS signal of a composition containing particles, it is possible to determine the cross-sectional radius of gyration ( _Rg ), and therefore the size of the particles, using an appropriate format (e.g., Zimm plot, Berry plot, or Debye plot). Preferably, the Zimm plot is calculated using the following formula (or its reciprocal):
where c is the mass concentration of the solvent (g/mL); A ₂ is the second virial coefficient (mol·mL/g ² ); P(θ) is a form factor related to the angular dependence of the scattered light intensity; R _θ is the excess Rayleigh ratio (cm ⁻¹ ); and K* is an optical constant equal to 4π ² η _o (dn/dc) ² λ ₀ ⁻⁴ N _A ⁻¹ , where η _o is the refractive index of the solvent at the incident radiation (vacuum) wavelength, λ ₀ is the incident radiation (vacuum) wavelength (nm), N _A is Avogadro's number (mol ⁻¹ ), and dn/dc is the differential refractive index concentration increment (mL/g).
(See, for example, Buchholz et al. (Electrophoresis 22 (2001), 4118-4128); B. H. Zimm (J. Chem. Phys. 13 (1945), 141; P. Debye (J. Appl. Phys. 15 (1944): 338; and W. Burchard (Anal. Chem. 75 (2003), 4279-4291). Preferably, the Berry plot is represented by the following terms or their reciprocals:
(wherein c, _Rθ and K* are as defined above.)
Preferably, the Debye plot is calculated as the following term or its reciprocal:
(wherein c, _Rθ and K* are as defined above.)
Calculate as follows.

As used herein, the phrase "dynamic light scattering" or "DLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of particles. A monochromatic light source, usually a laser, is projected onto a sample through a polarizer. The scattered light then passes through a second polarizer, where it is detected, and the resulting image is projected onto a screen. Particles in solution are struck by the light, causing it to diffract in all directions. The light diffracted from the particles interferes constructively (bright areas) or destructively (dark areas). This process is repeated at short intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity of each spot over time.

As used herein, the phrase "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's cross-sectional radius of gyration and/or particle molar mass. High-intensity monochromatic light, usually a laser, is directed into a solution containing the particles. One or more detectors are used to measure the scattered intensity at one or more angles. The angular dependence is necessary to obtain accurate measurements of both the molar mass and the size of the radius of the entire macromolecule. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), is generally considered the standard practice of static light scattering.

"Immunogenicity" is the ability of a foreign substance, such as RNA, to provoke an immune response in humans or other animals. The innate immune system is the component of the immune system that is relatively nonspecific and immediate. Along with the adaptive immune system, it is one of the two main components of the vertebrate immune system.

As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

As used herein, the term "exogenous" refers to any substance introduced into or produced outside of an organism, cell, tissue, or system.

The term "repeating unit" refers to a basic unit that repeats itself periodically along the polymer chain of a polymer and is derived from one monomer. The structures of a repeating unit and its corresponding monomer often coincide, but can differ from each other.

As used herein, the term "functionalized moiety" refers to a group of atoms in a molecule having a distinctive chemical property, where the atoms of the functionalized moiety are covalently bonded to each other and to the rest of the molecule. Preferably, the atoms of the functionalized moiety include at least one atom selected from the group consisting of O, N, and S. The functionalized moiety is monovalent (e.g., hydroxy, cyano, nitro, or amido (e.g., -C(O)NHCH ₃ )) or divalent (e.g., amide (e.g., —C(O)NH—), carbonyl (—C(O)—), or ester (e.g., —OC(O)—). In certain embodiments, the functionalizing moiety provides hydrophilicity to the group to which the functionalizing moiety is attached and/or at least one charge (positive or negative) to the group to which the functionalizing moiety is attached, e.g., by providing at least one hydrogen bond acceptor/donor. In certain embodiments, the functionalizing moiety is a hydrogen bond acceptor (e.g., a carbonyl moiety), a hydrogen bond donor (e.g., a hydroxyl moiety, —NH— (e.g., in an amide moiety) or a thiol moiety), or both (e.g., in an amide moiety) and/or a charge ( Examples of monovalent functionalized moieties include hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino (imine), imidothioate, thionylamide, carbonate, carbonitide, Examples of divalent functionalized moieties include ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide ... These include sulfite diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino(imine), imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

As used herein, particularly with respect to functionalized moieties as components of linkers, the term "hydroxyl" or "hydroxy" refers to the group -OH.

As used herein, the term "halogen" with respect to functionalized moieties, particularly as components of linkers, means fluoro, chloro, bromo, or iodo.

As used herein, particularly with respect to functionalized moieties as components of linkers, the term "cyano" refers to the group -CN.

As used herein, particularly with respect to functionalized moieties as components of linkers, the term "azido" refers to the group _N3 .

The term "nitro" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group _--NO.sub.2 .

The term "amino," as used herein with respect to functionalized moieties, particularly as components of linkers, includes unsubstituted amino (i.e., the group --NH ₂ ) and substituted amino (i.e., mono- or disubstituted amino in which one or two of the hydrogen atoms are replaced with a group other than hydrogen). Amino groups can be monovalent (e.g., --NRR, where each R is independently H or an organic group, such as R ⁷² or R ⁷³ , as defined below) or divalent (e.g., --NR--, where R is H or an organic group, such as R ⁷² , as defined below). In certain embodiments, the term "amino" refers to the group -N(R ⁷² )(R ⁷³ ), where R ⁷² and R ⁷³ at each occurrence are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ⁷² and R ⁷³ together with the nitrogen atom to which they are attached may form the group -N=CR ⁷⁵ R ⁷⁶ , where each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl. R ⁷⁵ and R 76 are independently substituted with one or more of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NH y R 80 2-y, or R 75 and R 76 are independently substituted with one or more of -NH y R 80 2-y (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); R ⁷⁵ and R ⁷⁶ are independently selected from the group consisting of -H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, and -NH _y R ⁸⁰ _2-y , or R ⁷⁵ and R ⁷⁶ together with the atoms to which they are attached can form a ring, which is optionally substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the ring, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected R ⁷⁰ , wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with an independently selected R ⁷⁰ (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); y is an integer from 0 to 2; R ⁸⁰ is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with an independently selected R ⁷⁰ (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1-5, 1-4, or 1-3, or 1 or 2); and R ⁷⁰ is other than H, preferably a first-level substituent, second-level substituent, or third-level substituent disclosed herein. In certain embodiments, each of R ⁷² and R ⁷³ is independently H or a hydrocarbyl group selected from the group consisting of H, C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl), wherein each of the hydrocarbyl groups (e.g., each of the C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl) groups) is optionally substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms bonded to the hydrocarbyl group (e.g., the C _1-6 alkyl, aryl, or aryl(C _1-6 alkyl) group)), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, for example, 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) independently selected R ⁷⁰ .

The term "ammonium" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to the group --N.sup. ⁺ ( ^R.sup.72 ) ^.sub.2 ( _R.sup.73 ), where ^R.sup.72 and ^R.sup.73 are as defined for the term "amino."

As used herein, particularly with respect to functionalized moieties as components of linkers, the terms "thiol" or "sulfanyl" refer to the group -SH.

As used herein, particularly with respect to functionalized moieties as components of linkers, the term "disulfanyl" refers to the group -SSH.

As used herein, particularly with respect to functionalized moieties as components of linkers, the term "carboxyl" or "carboxy" refers to the group -COOH.

The term "amide" or "amido" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a structure comprising the structure -C(O)NH- (including its isomeric arrangement, the structure -NHC(O)-, unless specified to the contrary). Preferably, each end of the amide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). (If both ends are bonded to the same organic group, the amide moiety also describes a lactam.) The amide group can be monovalent (e.g., -C(O)NRR or -NRC(O)R, where each R is H or an organic group such as one of the organic groups specified in the definition of ^R above in the definition of the term "amino") or divalent (e.g., -C(O)NR- or -NRC(O)-, where R is H or an organic group such as one of the organic groups specified in the definition of ^R above in the definition of the term "amino").

The term "ester," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to a group comprising the structure -C(O)O- (including its isomeric arrangements, the structure -OC(O)-, unless specified to the contrary). Preferably, each end of the ester structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). (If both ends are bonded to the same organic group, the ester moiety may also be referred to as a lactone.) The ester group can be monovalent (e.g., -C(O)OR or -OC(O)R, where R is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -C(O)O- or -OC(O)-).

The term "ether," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -O-, where each end of the ether structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The ether group can be monovalent (e.g., -OR, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -O-).

The term "sulfide" or "thioether," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -S-, where each end of the sulfide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfide group can be monovalent (e.g., -SR, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -S-).

The term "disulfide," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to a group comprising the structure -SS-, where each end of the disulfide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The disulfide group can be monovalent (e.g., -SSR, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -SS-).

The term "diselenide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SeSe-, where each end of the diselenide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The diselenide group can be monovalent (e.g., -SeSeR, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -SeSe-).

The term "sulfoxide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the sulfinyl structure -S(O)-, where each end of the sulfoxide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfoxide group can be monovalent (e.g., -S(O)R, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (as, e.g., -S(O)-).

The term "sulfone" as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the sulfonyl structure -S(O) ₂ -, where each end of the sulfone structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The sulfone group can be monovalent (e.g., -S(O) ₂ R, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., as in -S(O) ₂ -).

The term "sulfite," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OS(O)O-, where one of the two ends of the sulfite structure is covalently bonded to an atom of an organic group and the other end is covalently bonded to H or a C atom of the same or another organic group (e.g., an alkylene group as a further component of the linker). The sulfite group can be monovalent (e.g., -OS(O)OR, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -OS(O)O-).

The term "sulfate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OS(O) ₂ O-, where one of the two ends of the sulfate structure is covalently bonded to an atom of an organic group and the other end is covalently bonded to H or a C atom of the same or another organic group (e.g., an alkylene group as a further component of the linker). The sulfate group can be monovalent (e.g., -OS(O) ₂ OR, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -OS(O) ₂ O-).

The term "phosphate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OP(O)(OR)O-, where one of the two ends of the phosphate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The phosphate group can be monovalent (e.g., -OP(O)(OR) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OP(O)(OR)O-, where the term R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "sulfinamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -S(O)N(R)-, where the S-terminus of the sulfinamide is covalently bonded to a C-atom of an organic group (e.g., an alkylene group as a further component of the linker) and the N-terminus is covalently bonded to H or a C-atom of the same or another organic group (R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The sulfinamide group can be monovalent (e.g., -S(O)N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -S(O)N(R)-, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "sulfonamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -S(O) _2N (R)-, where the S-terminus of the sulfonamide is covalently bonded to a C-atom of an organic group (e.g., an alkylene group as a further component of the linker) and the N-terminus is covalently bonded to H or a C-atom of the same or another organic group (R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The sulfonamide group can be monovalent (e.g., -S(O) _2N (R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -S(O) _2N (R)-, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "sulfamate", as used herein with respect to a functionalized moiety, particularly as a component of a linker, refers to a group comprising the structure -OS(O) ₂ N(R)- (including its isomeric arrangement, -N(R)S(O) ₂ O-, unless specified to the contrary), in which one end of the sulfamate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino"). The sulfamate group can be monovalent (e.g., —OS(O) ₂ N(R) ₂ or —N(R)S(O) ₂ OR, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —OS(O) ₂ N(R)— or —N(R)S(O) ₂ O—, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "sulfite diamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)S(O)N(R)-, where one end of the sulfite diamide structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The sulfite diamide group can be monovalent (e.g., -N(R)S(O)N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -N(R)S(O)N(R)-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "acidous diamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)S(O) _2N (R)-, where one end of the acidous diamide structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino"). The acid diamide group can be monovalent (e.g., —N(R)S(O) ₂ N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —N(R)S(O) ₂ N(R)—, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "urea," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(O)N(R)-, where one end of the urea structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The urea group can be monovalent (e.g., -N(R)C(O)N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -N(R)C(O)N(R)-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "thiourea," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(S)N(R)-, where one end of the thiourea structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of a linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The thiourea group can be monovalent (e.g., -N(R)C(S)N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -N(R)C(S)N(R)-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbonyl," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)-, where one end of the carbonyl structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (if both ends are bonded to C atoms of organic groups, the carbonyl moiety is also referred to as a "keto" moiety). The carbonyl group can be monovalent (e.g., -C(O)R, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -C(O)-).

The term "thiocarbonyl," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(S)-, where one end of the thiocarbonyl structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group. The thiocarbonyl group can be monovalent (e.g., -C(S)R, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -C(S)-).

The term "orthoester," as used herein, particularly with respect to functionalized moieties as components of linkers, refers to a moiety that includes a C atom bonded with three alkoxy groups (i.e., -OR, where R is an organic group (e.g., an alkylene group as a further component of a linker), such as one of the organic groups specified in the definition of R given ^above in the definition of the term "amino." Exemplary formulas of orthoesters include the structure (-O) _rC (OR) _3-r- , where each R is independently an organic group, such as an organic group independently selected from the organic groups specified in the definition of R given above in the definition of the term " ^amino ." r is 1 or 2; and each end of the orthoester structure is covalently bonded to a C atom of an additional organic group or two further organic groups. In certain embodiments, the orthoester comprises the structure (—O) _r C(OR ²⁵ ) _3-r —, where each R ²⁵ is independently a hydrocarbyl group such as C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl), which is optionally substituted (e.g., with one or more first-level, second-level, or third-level substituents as defined herein); r is 1 or 2; and each end of the orthoester structure is covalently bonded to a C atom of an additional organic group or two further distant organic groups. The orthoester group can be monovalent (e.g., —C(OR) ₃ or —OC(OR) ₂ R, where each R is an organic group such as independently selected from the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., (—O) ₂ C(OR)(R) or —OC(OR) ₂ —, where each R is an organic group such as independently selected from the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "thioate" or "thioester," as used herein with respect to a functionalized moiety, particularly as a component of a linker, refers to a group comprising the structure -SC(O)- (including, unless specified to the contrary, its isomeric arrangements -C(O)S-, -OC(S)-, and -C(S)O-), where each end of the thioate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The thioate group can be monovalent (e.g., -SC(O)R or -C(O)SR or -OC(S)R or -C(S)OR, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(O)- or -C(O)S- or -OC(S)- or -C(S)O-).

The term "dithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)- (including its isomeric arrangement, -C(S)S-, unless specified to the contrary), where each end of the dithioate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The dithioate group can be monovalent (e.g., -SC(S)R or -C(S)SR, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(S)- or -C(S)S-).

The term "imidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)- (including its isomeric arrangements of the structure -C(=NR)O-, unless specified to the contrary), where each end of the imidate structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker), where each R is independently H or an organic group such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino." Imidate groups can be monovalent (e.g., -OC(=NR)R' or -C(=NR)OR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and each R' is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(=NR)- or -C(=NR)O-, where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "imino" or "imine," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(=NR)-, where one end of the imino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The moiety -C(=NR)H is also known as an "aldimine," and the moiety -C(=NR)R' (where R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") is also known as a "ketimine." An imino group can be monovalent (e.g., -C(=NR)R, where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and each R' is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -C(=NR)-, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "imidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(=NR)S- (including its isomeric arrangement, -SC(=NR)-, unless specified to the contrary), wherein one end of the imidothioate structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group, such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"). The imidothioate group can be monovalent (e.g., -C(=NR)SR or -SC(=NR)R, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -C(=NR)S- or -SC(=NR)-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "thionylamino," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(S)NR- (including its isomeric arrangements, -N(R)C(S)-, unless specified to the contrary), wherein one end of the thionylamino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group (where each R is independently H or an organic group, such as one of the organic groups specified in the definition of R given above in the definition of the term " ^amino "). A thionylamino group can be monovalent (e.g., -C(S)NRR or -N(R)C(S)R, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -C(S)NR- or -N(R)C(S)-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbonate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(O)O-, where each end of the carbonate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonate group can be monovalent (e.g., -OC(O)OR', where R' is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -OC(O)O-).

The term "carbonothioate," as used herein with respect to a functionalized moiety, particularly as a component of a linker, refers to a group containing the structure -OC(S)O- or -OC(O)S- (including the isomeric arrangements thereof -SC(O)O-, unless specified to the contrary), in which each end of the carbonothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonothioate group can be monovalent (e.g., -OC(S)OR' or -OC(O)SR' or -SC(O)OR', where each R' is independently an organic group, such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(S)O- or -OC(O)S- or -SC(O)O-).

The term "carbonodithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group containing the structure -SC(O)S- or -OC(S)S- (including its isomeric arrangements of the structure -SC(S)O-, unless specified to the contrary), in which each end of the carbonodithioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonodithioate group can be monovalent (e.g., -SC(O)SR'-OC(S)SR' or -SC(S)OR', where each R' is independently an organic group, such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(O)S- or -OC(S)S- or -SC(S)O-).

The term "carbonotrithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)S-, where each end of the carbonotrithioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker). The carbonotrithioate group can be monovalent (e.g., -SC(S)SR, where R is an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., -SC(S)S-).

The term "guanidino" or "imidamide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(=NR)NR- (where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"), where one end of the guanidino structure is covalently bonded to an organic group (e.g., an alkylene group as a further component of the linker) and the other end is covalently bonded to H or a C atom of the same or another organic group. The guanidino group can be monovalent (e.g., -N(R)C(=NR)NR-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -N(R)C(=NR)NR-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbamidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)NR- (including its isomeric arrangements, unless specified to the contrary, of the structure -N(R)C(=NR)O-), in which the O terminus of the carbamidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other (N) terminus is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of R ^given above in the definition of the term "amino"). Carbamimidate groups can be monovalent (e.g., -OC(=NR)NRR or -N(R)C(=NR)OR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(=NR)NR- or -N(R)C(=NR)O-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbonimidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)O-, where each end of the carbonimidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The carbonimidate group can be monovalent (e.g., -OC(=NR)OR', where R is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(=NR)O-, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbamate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(O)NR- (including its isomeric arrangements, -N(R)C(O)O-, unless specified to the contrary), where the O-terminus of the carbamate structure is covalently bonded to a C-atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C-atom of the same or another organic group (where each R is independently H or an organic group, such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"). Carbamate groups can be monovalent (e.g., -OC(O)NRR or -N(R)C(O)OR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(O)NR- or -N(R)C(O)O-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbamodithioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(S)NR- (including its isomeric arrangements -N(R)C(S)S-, unless specified to the contrary), in which the S-terminus of the carbamodithioate is covalently bonded to a C-atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C-atom of the same or another organic group (each R is independently H or an organic group such as one of the organic groups specified in the definition of R given above in ^the definition of the term "amino"). Carbamodithioate groups can be monovalent (e.g., -SC(S)NRR or -N(R)C(S)SR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(S)NR- or -N(R)C(S)S-, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbonodithioimidate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(=NR)S-, where each end of the carbonodithioimidate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) (each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"). The carbonodithioimidate group can be monovalent (e.g., -SC(=NR)SR', where R is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(=NR)S-, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbamimidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -SC(=NR)NR- (including its isomeric arrangements, -N(R)C(=NR)S-, unless specified to the contrary), in which the S-terminus of the carbamimidothioate is covalently bonded to a C-atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C-atom of the same or another organic group (each R is independently H or an organic group, such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"). The carbamimidothioate group can be monovalent (e.g., -SC(=NR)NRR or -N(R)C(=NR)SR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -SC(=NR)NR- or -N(R)C(=NR)S-, where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "carbamothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)C(O)S- or -N(R)C(S)O- (including, unless specified to the contrary, the isomeric arrangements thereof -SC(O)NR- or -OC(S)NR-), wherein the O/S terminus of the carbamothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to a C atom of the same or another organic group (each R is independently H or an organic group such as one of the organic groups specified in the definition of R given above in the definition of the term " ^amino "). A carbamothioate group can be monovalent (e.g., —N(R)C(O)SR′ or —N(R)C(S)OR′ or —SC(O)NRR or —OC(S)NRR, where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”; and each R′ is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —N(R)C(O)S— or —N(R)C(S)O— or —SC(O)NR— or —OC(S)NR—, where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "carbonimidothioate," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(=NR)S- (including its isomeric arrangement, the structure -SC(=NR)O-, unless specified to the contrary), in which the O/S terminus of the carbonimidothioate structure is covalently bonded to a C atom of an organic group (e.g., an alkylene group as a further component of the linker) and the other end (N-terminus) is covalently bonded to H or a C atom of the same or another organic group (each R is independently H or an organic group such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"). The carbonimidothioate group can be monovalent (e.g., -OC(=NR)SR' or -SC(=NR)OR', where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino"; and each R' is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(=NR)S- or -SC(=NR)O-, where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "acylhydrazone," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(R')(=N-N(R)C(O)-) (including its isomeric arrangements (-C(O)(N(R)-N=)C(R')-) unless specified to the contrary) and/or =C(=N-N(R)C(O)R'), where each R' is independently an organic group such as one of the organic groups specified in the ^R definition set forth above in the definition of the term "amino"; each R is H or an organic group such as one of the organic groups specified in the ^R definition set forth above in the definition of the term "amino"; and each end of the acylhydrazone structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of a linker). In certain embodiments, an acylhydrazone has the structure -C( ^R )(=N-N( ^R )C(O)—) (including isomeric arrangements thereof (including —C(O)(N(R ²⁶ )—N═)C(R ²⁵ )—) and/or ═C(═N—N(R ²⁶ )C(O)R ²⁵ ), unless specified to the contrary, where each R ²⁵ is independently a hydrocarbyl group such as C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl), which is optionally substituted (e.g., with one or more first level substituents, second level substituents, or third level substituents as defined herein); each R ²⁶ is independently H or C _1-6 alkyl, aryl, and aryl(C and a hydrocarbyl group such as _1-6 alkyl), which is optionally substituted (e.g., with one or more of the first-level, second-level, or third-level substituents defined herein); and each of the ends of the acylhydrazone structure is covalently bonded to a C atom of an additional organic group or two further apart organic groups. An exemplary chemical structure of an acylhydrazone is shown below:
[During the ceremony,
represents the bond covalently linking the acylhydrazone to a further organic group (e.g., an alkylene group as a further component of the linker).
An acylhydrazone group can be monovalent (e.g., —C(R′)(═N—N(R)C(O)R′) or —C(O)(N(R)—N═)C(R′) ₂ , where each R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”; and each R′ is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —C(R′)(═N—N(R)C(O)—, —C(O)(N(R)—N═)C(R′)—, or ═C(═N—N(R ²⁶ )C(O)R ²⁵ , where each R′ is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”; and each R′ is independently H or an organic group such as one of the organic groups specified in the definition of R 72 set forth above in the definition of the term “amino”). ⁷²⁾基团。 72 may be an organic group such as one of the organic groups specified in the definition.

The term "hydrazine," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -N(R)N(R)-, where each R is independently H or an organic group such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"; and each end of the hydrazine structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as further components of the linker). In certain embodiments, the hydrazine comprises the structure -N( ^R )N( ^R )-, where each ^R is independently H or a hydrocarbyl group such as C _alkyl , aryl, and aryl(C _alkyl ), which is optionally substituted (e.g., with one or more first-level substituents, second-level substituents, or third-level substituents as defined herein); and each end of the hydrazine structure is covalently bonded to a C atom of an additional organic group or two further organic groups. The hydrazine group can be monovalent (e.g., —N(R)N(R) ₂ , where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —N(R)N(R)—, where each R′ is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "oxime," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group that includes the structure =C(=N(OH)), where each end of the oxime structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). An exemplary chemical formula of an oxime is shown below:
[During the ceremony,
represents the bond covalently linking the oxime to an additional organic group.
The oxime group can be monovalent (e.g., -C(=N(OH))(R), where each R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² given above in the definition of the term "amino") or divalent (e.g., =C(=N(OH))).

The term "acetal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OCH(R')O-, where R' is an organic group such as one of the organic groups specified in the definition of ^R given above in the definition of the term "amino"; and each of both O atoms of the acetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as further components of the linker). In certain embodiments, the acetal comprises the structure -OCH(R ²⁵ )O-, where R ²⁵ is a hydrocarbyl group such as C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl), which is optionally substituted (e.g., with one or more first-level substituents, second-level substituents, or third-level substituents as defined herein); and each of both O atoms of the acetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups. The acetal group can be monovalent (e.g., -OCH(R')OR', where each R' is independently an organic group such as independently selected from the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OCH(R')O-, where R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "hemiacetal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OCH(OH)-, where each end of the hemiacetal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as additional components of the linker). The hemiacetal group can be monovalent (e.g., -OCH(OH)OR', where R' is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OCH(OH)-).

The term "ketal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OC(R')(R')O-, where each R' is independently an organic group, such as independently selected from the organic groups identified in the definition of ^R given above in the definition of the term "amino," and each of both O atoms of the ketal structure is covalently bonded to a C atom of an additional organic group or two further organic groups (e.g., alkylene groups as further components of the linker). In certain embodiments, the ketal comprises the structure -OC( ^R25 )( ^R25 )O-, where each ^R25 is independently a hydrocarbyl group such as _C1-6 alkyl, aryl, and aryl( _C1-6 alkyl), which is optionally substituted (e.g., with one or more first-level, second-level, or third-level substituents as defined herein); and each of both O atoms of the ketal structure is covalently bonded to a C atom of an additional organic group or two further organic groups. The ketal group can be monovalent (e.g., -OC(R')(R')OR', where each R' is independently an organic group such as an organic group independently selected from those specified in the definition of R ⁷² set forth above in the definition of the term "amino") or divalent (e.g., -OC(R')(R')O-, where each R' is independently an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term "amino").

The term "hemiketal," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -OCR'(OH)-, where R' is an organic group such as one of the organic groups identified in the definition of ^R provided above in the definition of the term "amino," and each end of the hemiketal structure is covalently bonded to a C-atom of an additional organic group or two further organic groups (e.g., alkylene groups as further components of the linker). In certain embodiments, the hemiketal comprises the structure ^-OCR25 (OH)-, where ^R25 is a hydrocarbyl group such as _C1-6 alkyl, aryl, and aryl( _C1-6 alkyl), which is optionally substituted (e.g., with one or more first-level, second-level, or third-level substituents as defined herein); and each end of the hemiketal structure is covalently bonded to a C-atom of an additional organic group or two further organic groups. The hemiketal group can be monovalent (e.g., —OC(R′) ₂ (OH), where each R′ is independently an organic group such as an organic group independently selected from those specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —OCR′(OH)—, where R′ is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

The term "imide," as used herein, particularly with respect to a functionalized moiety as a component of a linker, refers to a group comprising the structure -C(O)N(R)C(O)-, where R is an organic group such as one of the organic groups specified in the definition of R given above in the definition of the term "amino ^" ; and each end of the imide structure is covalently bonded to a C atom of the same organic group or two separate organic groups (e.g., alkylene groups as further components of the linker). The imido group can be monovalent (e.g., —C(O)N(R)C(O)R′, where R is independently H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”; and R′ is an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”) or divalent (e.g., —C(O)N(R)C(O)—, where R is H or an organic group such as one of the organic groups specified in the definition of R ⁷² set forth above in the definition of the term “amino”).

As used herein in the context of organic groups, the term "acyclic" refers to an open-chain organic group that does not contain a ring. An "open-chain" or "acyclic" organic group can be straight-chained (i.e., containing only one unbranched chain without any side chains) or branched (i.e., the main chain "contains" one or more side chains).

An organic group that is "substituted with one or more substituents" means that one or more (e.g., from 1 up to the maximum number of hydrogen atoms bonded to the organic group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, e.g., 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the organic group have been replaced with something other than hydrogen (when more than one hydrogen atom is replaced, the substituents can be the same or different). Preferably, the one or more substituents can be selected from the first-level substituents, second-level substituents, or third-level substituents described herein.

As used herein, the phrase "hydrogen bond" or "H-bond" refers to a non-covalent bond (in some embodiments, primarily due to electrostatic attraction) between (i) a hydrogen atom covalently bonded to a more electronegative atom or group and (ii) a lone pair of electrons on another electronegative atom. In some embodiments, the more electronegative atom or group includes nitrogen and oxygen atoms; thus, examples of groups in which a hydrogen atom is covalently bonded to a more electronegative atom or group include amino groups bearing at least one covalently bonded hydrogen atom, the -NH- group of an amide group, hydroxyl groups (e.g., as in alcohols) or as part of other functional groups (e.g., as part of a carboxyl (-COOH) group), and sulfanyl groups (e.g., as in thiols) or as part of other functional groups (e.g., as part of a disulfanyl (-SSH) or thioester (-C(OSH)) group). In some embodiments, the lone pair of electrons of the other electronegative atom is the lone pair of an oxygen atom present in a carbonyl group or the lone pair of a nitrogen atom present in a primary, secondary, or tertiary amino group.

As used herein, the phrase "hydrogen bond donor" refers to an atomic, ionic, or molecular component of a hydrogen bond that provides a bridging (covalent) hydrogen atom. In certain embodiments, hydrogen bond donors include amino groups bearing at least one covalently bonded hydrogen atom, -NH- groups of amide groups, hydroxyl groups (e.g., as in alcohols) or as part of other functional groups (e.g., as part of a carboxyl (-COOH) group), and sulfanyl groups (e.g., as in thiols) or as part of other functional groups (e.g., as part of a disulfanyl (-SSH) or thioester (-C(OSH)) group).

As used herein, the phrase "hydrogen bond acceptor" refers to an atomic, ionic, or molecular component of a hydrogen bond that does not donate a bridging (covalent) hydrogen atom. In some embodiments, the hydrogen bond acceptor contains at least one lone pair of electrons. Examples of hydrogen bond acceptors include carbonyl moieties and primary, secondary, and tertiary amino groups.

The term "stealth" as used herein refers to the ability of the particles described herein to be detected, then sequestered and/or degraded, or to be barely detected, then sequestered and/or degraded, and/or to be subsequently detected, then sequestered and/or degraded, by the immune system of the host to which they are administered.

The term "phosphatidylethanolamine" refers to a compound having the following formula:
or a salt thereof, wherein in each case, acyl refers to an acyl moiety (e.g., a —C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably straight-chained). In certain embodiments, each acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moieties may be saturated or unsaturated (e.g., monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In certain embodiments, one acyl is saturated and the other is unsaturated (e.g., monounsaturated). Examples of acyl moieties include -C(O)( _CH2 ) _{16CH3 (} stearoyl), -C(O)( _CH2 ) _14CH3 (palmitoyl), -C(O)( _CH2 ) _{12CH3 (} myristoyl), and -cis-C(O)( _CH2 ) ₇ -CH=CH-( _CH2 ) _7CH3 (oleoyl). The term "phosphatidylethanolamine moiety" means a monovalent radical of phosphatidylethanolamine, preferably _one in which _the hydrogen atom of the amino group has been removed.

The term "DSPE" refers to a compound of the formula:
or a salt thereof, wherein in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl). The term "distearoylphosphatidylethanolamine moiety" means a monovalent radical of DSPE, preferably one in which the hydrogen atom of the amino group has been removed.

The term "DPPE" refers to a compound of the formula:
or a salt thereof, wherein in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl). The term "dipalmitoylphosphatidylethanolamine moiety" means a monovalent radical of DPPE, preferably one in which the hydrogen atom of the amino group has been removed.

The term "DOPE" refers to a compound of the formula:
or a salt thereof, wherein in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl). The term "dioleoylphosphatidylethanolamine moiety" means a monovalent radical of DOPE, preferably one in which the hydrogen atom of the amino group has been removed.

The term "POPE" refers to a compound of the formula:
or a salt thereof, wherein -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl); and -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl). The term "palmitoyloleoylphosphatidylethanolamine moiety" refers to a monovalent radical of POPE, preferably one in which the hydrogen atom of the amino group has been removed.

The term "tocopherol" refers to a compound of the formula:
where each of R _t1 and R _t2 is independently H or methyl. In α-tocopherol, R t1 and R t2 are both methyl; in β-tocopherol, R _t1 is methyl and R _t2 is H; in γ-tocopherol, R _{t1 is H and R t2 is} methyl; and in _{δ-tocopherol, R t1 and} R _t2 are both H. The term "tocopherol moiety" or "tocopheryl moiety" refers to a monovalent radical of tocopherol, preferably one _in which the hydrogen atom of a hydroxy group has been removed.

The term "DAG" refers to a group having the formula:
or a salt thereof, wherein in each case acyl refers to an acyl moiety (e.g., a —C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably straight-chain). In certain embodiments, each acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moieties may be saturated or unsaturated (e.g., monounsaturated). Thus, both acyl moieties may be saturated or unsaturated (e.g., monounsaturated). In certain embodiments, one acyl is saturated and the other is unsaturated (e.g., monounsaturated). Examples of acyl moieties include -C(O)( _CH2 ) _{16CH3 (} stearoyl), -C(O)( _CH2 ) _14CH3 (palmitoyl), -C(O)( _CH2 ) _{12CH3 (} myristoyl), and -cis-C(O)( _CH2 ) ₇ -CH=CH-( _CH2 ) _7CH3 ( _oleoyl ). For example _, DMG refers to 1,2-dimyristoylglycerol, i.e., a diacylglyceride of the above formula in which both acyl groups are _{-C(O)(CH2)12CH3 ( myristoyl} ). The term "diacylglyceride moiety" refers to a monovalent radical of a diacylglyceride, preferably one in which the hydrogen atom of a hydroxy group has been removed.

The term "DAA" refers to a dialkylamine having the formula HN(alkyl) ₂ or a salt thereof, where each alkyl moiety is preferably linear. In certain embodiments, each alkyl moiety has at least 8 carbon atoms. Preferably, each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl moieties _include -( _CH2 ) _17CH3 (stearyl), - _{( CH2} ) _15CH3 (palmityl), and -( _CH2 ) _13CH3 (myristyl). For example, DMA refers to 1,2-dimyristylamine, i.e., a dialkylamine of the above formula in which both _alkyl groups are -( _CH2 ) _13CH3 (myristyl). The term "dialkylamine moiety" refers to a monovalent radical of a dialkylamine, preferably one _in which a hydrogen atom of an amino group has been removed.

The term "ceramide" refers to a compound having the formula:
or a salt thereof, wherein —C ₁₃ H ₂₇ refers to the moiety —(CH ₂ ) ₁₂ CH ₃ ; and acyl refers to an acyl moiety (e.g., a —C(O)-hydrocarbyl moiety, where the hydrocarbyl group is preferably straight-chain). In certain embodiments, the acyl moiety is the acyl moiety of a fatty acid, more preferably the acyl moiety of a fatty acid having at least 8 carbon atoms. The acyl moiety may be saturated or unsaturated (e.g., monounsaturated). Examples of acyl moieties include —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), —C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), —C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), and —cis-C(O)(CH ₂ ) ₇ —CH═CH—(CH ₂ ) ₇ CH ₃ (oleoyl). For example, palmitoylceramide refers to a ceramide of the above formula in which the acyl is _-C (O)( _CH2 ) _14CH3 (palmitoyl). The term "ceramide moiety" refers to a monovalent radical of ceramide, preferably one in which the hydrogen atom of a hydroxy group (preferably the hydrogen of the terminal (primary) hydroxy group) has been removed.

The term "MAA" refers to a monoalkylamine having the formula H ₂ N(alkyl), or a salt thereof, where the alkyl moiety is preferably linear. In certain embodiments, the alkyl moiety has at least 8 carbon atoms. Preferably, the alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably the alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms. Examples of alkyl include -(CH ₂ ) ₁₇ CH ₃ (stearyl), -(CH ₂ ) ₁₅ CH ₃ (palmityl), and -(CH ₂ ) ₁₃ CH ₃ (myristyl). For example, MMA refers to myristylamine, i.e., a monoalkylamine of the above formula in which the alkyl group is -(CH ₂ ) ₁₃ CH ₃ (myristyl). The term "monoalkylamine moiety" refers to a monovalent radical of a monoalkylamine, preferably one in which one of the hydrogen atoms of the amino group has been removed.

Nucleic Acids The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term includes genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. Nucleic acids can exist as single-stranded or double-stranded and linear or covalently closed circular molecules. Nucleic acids can be isolated. The term "isolated nucleic acid," according to the present invention, means that the nucleic acid has been (i) amplified in vitro, e.g., by polymerase chain reaction (PCR) for DNA or in vitro transcription (e.g., using RNA polymerase) for RNA; (ii) recombinantly produced by cloning; (iii) purified, e.g., by cleavage and separation by gel electrophoresis; or (iv) synthesized, e.g., by chemical synthesis.

The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be thought of as a nucleotide without the phosphate group. A nucleoside is a nucleic acid base linked to a sugar (e.g., ribose or deoxyribose), while a nucleotide consists of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

The five standard nucleosides that commonly make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated to the single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly referred to as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA), but not ribonucleic acid (RNA). Conversely, uridine is found in RNA, but not DNA. The remaining three nucleosides may be found in both RNA and DNA therapies. In RNA, they are represented as A, C, and G, while in DNA, they are represented as dA, dC, and dG.

The modified purine (A or G) or pyrimidine (C, T or U) base moiety is preferably modified by one or more alkyl groups, more preferably one or more C _1-4 alkyl groups, and even more preferably one or more methyl groups. Particular examples of modified purine or pyrimidine base moieties include N ^-alkyl -guanine, N -alkyl-adenine, ⁵ -alkyl-cytosine, 5-alkyl-uracil and N(1)-alkyl-uracil, such as N ^-C _1-4 alkyl-guanine, N -C _1-4 alkyl-adenine, ⁵ -C _1-4 alkyl-cytosine, 5-C _1-4 alkyl-uracil and N(1)-C _1-4 alkyl-uracil, preferably N ^-methyl -guanine, N ^-methyl -adenine, 5-methyl-cytosine, 5-methyl-uracil and N(1)-methyl-uracil.

In some embodiments of all aspects of the invention, the nucleic acid is DNA.

As used herein, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA, e.g., partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and/or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the ends of the DNA. It is also contemplated herein that the nucleotides of DNA may be chemically synthesized nucleotides or non-standard nucleotides, such as ribonucleotides. For purposes of the present invention, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, particularly cDNA. cDNA may be obtained by reverse transcription of RNA.

RNA
In some embodiments of all aspects of the invention, the nucleic acid is RNA.

According to the present invention, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA comprises all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the termini of the RNA. It is also contemplated herein that the nucleotides of the RNA may be chemically synthesized nucleotides or non-standard nucleotides, such as deoxynucleotides. For purposes of the present invention, these altered/modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNA containing such altered/modified nucleotides or nucleosides (i.e., altered/modified RNA) can be referred to as an analog of naturally occurring RNA. A molecule contains "majority ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (regardless of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or their analogs).

"RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (e.g., antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activator RNA (e.g., small activator RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide, or protein. The RNA can express the encoded peptide, polypeptide, or protein. For example, the RNA can be RNA that encodes and expresses a pharmaceutically active peptide or protein. In some embodiments, the RNA can interact with the cellular translation machinery to enable translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), secrete the encoded peptide or protein, or produce it on its surface. Alternatively, the RNA can be non-coding RNA, such as antisense RNA, microRNA (miRNA), or siRNA.

As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT does not use live/cultured cells, but rather uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)).

mRNA
In some embodiments of all aspects of the invention, the nucleic acid is mRNA.

According to the present invention, the term "mRNA" refers to "messenger RNA" and includes "transcripts" that can be produced using a DNA template. Generally, mRNA encodes a peptide, polypeptide, or protein. Typically, mRNA comprises a 5' UTR, a peptide/protein coding region, and a 3' UTR. In the present invention, mRNA is preferably produced from a DNA template by in vitro transcription (IVT). As noted above, in vitro transcription methods are known to those skilled in the art, and a variety of in vitro transcription kits are commercially available.

Although mRNA is single-stranded, it may contain self-complementary sequences that allow it to fold back on itself and pair with itself to form a double helix.

According to the present invention, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

In a preferred embodiment of the present invention, mRNA refers to an RNA transcript that encodes a peptide, polypeptide, or protein.

In one embodiment, preferably the RNA encoding the peptide, polypeptide, or protein has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

As is well-established in the art, RNA (e.g., mRNA) generally comprises a 5' untranslated region (5' UTR), a peptide/polypeptide/protein coding region, and a 3' untranslated region (3' UTR). In some embodiments, RNA (e.g., mRNA) is produced by in vitro transcription or chemical synthesis. In some embodiments, RNA (e.g., mRNA) is produced by in vitro transcription using a DNA template. In vitro transcription methods are known to those skilled in the art, see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Additionally, a variety of in vitro transcription kits are commercially available, for example, from Thermo Fisher Scientific (e.g., Transcription Aid ^™ T7 Kit, MEGAscript® T7 Kit, MAXIscript®), New England BioLabs Inc. (e.g., HiScribe ^™ T7 Kit, HiScribe ^™ T7 ARCA mRNA Kit), Promega (e.g., RiboMAX ^™ , HeLaScribe®, Riboprobe® Systems), Jena Bioscience (e.g., SP6 or T7 Transcription Kit), and Epicentre (e.g., AmpliScribe ^™ ). To refer to modified RNA (e.g., mRNA), correspondingly modified nucleotides, e.g., modified naturally occurring nucleotides, non-naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription) or modifications can be made and/or added to mRNA after transcription.

In one embodiment, the RNA (e.g., mRNA) is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In certain embodiments of the present invention, the RNA (e.g., mRNA) is a "replicon RNA" (e.g., a "replicon mRNA") or simply a "replicon," particularly a "self-replicating RNA" (e.g., a "self-replicating mRNA") or a "self-amplifying RNA" (or "self-amplifying mRNA"). In certain embodiments, the replicon or self-replicating RNA (e.g., a self-replicating mRNA) is derived from or includes elements from an ssRN virus, particularly a positive-strand sRN virus such as an alphavirus. Alphaviruses are a typical example of a positive-strand RN virus. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) and structural proteins (virion formation). There are typically two open reading frames (ORFs) in the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded by a first ORF that together initiates near the 5' end of the genome, while the alphavirus structural proteins are encoded by a second ORF that together is found downstream of the first ORF and extends near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, roughly in a 2:1 ratio. In cells infected with alphaviruses, only the nucleic acid sequences encoding nonstructural proteins are translated from genomic RNA, while the genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules that mimic eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts like messenger RNA to directly translate the open reading frame encoding the nonstructural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or target organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced with an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems utilize alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated in trans by the replicase (hence the name "trans-replication system"). Trans-replication requires the presence of both nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated in trans by the replicase must contain certain alphavirus sequence elements to allow recognition and RNA synthesis by the alphavirus replicase.

In certain embodiments of the invention, an RNA (e.g., mRNA) described herein (e.g., included in a composition and/or used in a method of the invention) contains one or more modifications, e.g., to increase stability and/or translation efficiency and/or decrease cytotoxicity. For example, to increase expression of an RNA (e.g., mRNA), modifications can be made within the coding region, i.e., the sequence encoding the expressed peptide or protein, preferably without altering the sequence of the expressed peptide or protein. Such modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include: 5' cap structures; extension or shortening of a naturally occurring poly(A) tail; alteration of the 5' and/or 3' untranslated region (UTR), such as the introduction of a UTR unrelated to the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the G/C content of the RNA). According to the present invention, the term "modified" in the context of modified mRNA preferably relates to any modification of the mRNA that does not naturally occur in the RNA (e.g., mRNA).

In some embodiments, an RNA (e.g., an mRNA) described herein comprises a 5' cap structure. In some embodiments, an mRNA does not have an uncapped 5'-triphosphate. In some embodiments, an RNA (e.g., an mRNA) described herein comprises a conventional 5' cap and/or a 5' cap analog. The term "conventional 5'cap" refers to the cap structure found at the 5' end of an mRNA molecule, generally consisting of guanosine 5'-triphosphate (Gppp) attached by a triphosphate moiety to the 5' end of the next nucleotide in the mRNA (i.e., the guanosine is attached to the remainder of the mRNA via a 5'-5' triphosphate linkage). The guanosine may be methylated at the ^N7 position (resulting in the cap structure ^m7Gppp ). The term "5' cap analog" refers to a 5' cap that is based on a conventional 5' cap but is modified at the 2' or 3' position of the ^m7 guanosine structure to avoid incorporation of the 5' cap analog in the reverse orientation (such 5' cap analogs are also referred to as anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are those having one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as the phosphorothioate-modified 5' cap analog of the β-phosphate described in PCT/EP2019/ ⁰⁵⁶⁵⁰² (e.g., _m27,2'OG (5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA)). Addition of a 5' cap structure as described herein to RNA (e.g., mRNA) can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, where the 5' cap structure is co-transcriptionally incorporated into the RNA (e.g., mRNA) strand produced, or RNA (e.g., mRNA) can be produced, for example, by in vitro transcription, and the 5' cap structure can be post-transcriptionally attached to the mRNA using a capping enzyme, e.g., vaccinia virus capping enzyme.

In some embodiments, the RNA (e.g., mRNA) comprises a 5' cap structure selected from the group consisting of m ₂ ^7,2'O G(5')ppSp(5')G (particularly its D1 diastereomer), m ₂ ^7,3'O G(5')ppp(5')G, and m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG. In some embodiments, the RNA encoding a peptide, polypeptide, or protein comprising an antigen or epitope comprises m ₂ ^7,2'O G(5')ppSp(5')G (particularly its D1 diastereomer) as the 5' cap structure.

In some embodiments, RNA (e.g., mRNA) comprises cap 0, cap 1, or cap 2, preferably cap 1 or cap 2. According to the present invention, the term "cap 0" refers to the structure "m ⁷ GpppN," where N is any nucleoside bearing an OH moiety at the 2' position. According to the present invention, the term "cap 1" refers to the structure "m ⁷ GpppNm," where Nm is any nucleoside bearing an _OCH3 moiety at the 2' position. According to the present invention, the term "cap 2" refers to the structure "m ⁷ GpppNmNm," where each Nm independently is any nucleoside bearing an _OCH3 moiety at the 2' position.

The 5' cap analog beta-S-ARCA (β-S-ARCA) has the following structure:

The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA (D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column, and therefore has a shorter retention time, compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA (D2)). The HPLC is preferably analytical HPLC. In one embodiment, a Supelcosil LC-18-T RP column, preferably in a 5 μm, 4.6 x 250 mm format, is used for the separation, where a flow rate of 1.3 ml/min can be applied. In one embodiment, a gradient of methanol in ammonium acetate is used, e.g., a 0-25% linear gradient of methanol in 0.05 M ammonium acetate within 15 minutes. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

The 5' cap analog m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG (also referred to as m ₂ ^7,3'O G(5')ppp(5')m ^2'-O ApG), a component of Cap 1, has the following structure:

An example of a cap 0 mRNA containing β-S-ARCA and mRNA has the following structure:

An example of a cap 0 mRNA containing m ₂ ^7,3'O G(5')ppp(5')G and mRNA has the following structure:

An example of a cap 1 mRNA containing m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG and mRNA has the following structure:

In some embodiments, an RNA (e.g., an mRNA) comprises a 3'-poly(A) sequence. As used herein, the term "poly(A) tail" or "poly(A) sequence" refers to an uninterrupted or interrupted sequence of adenylic acid residues typically located at the 3' end of an RNA (e.g., an mRNA) molecule. Poly(A) tails or poly(A) sequences are known to those of skill in the art and may follow an RNA (e.g., an mRNA) described herein in the 3' UTR. An uninterrupted poly(A) tail is characterized by consecutive adenylic acid residues. In nature, an uninterrupted poly(A) tail is typical. The RNA (e.g., an mRNA) disclosed herein may have a poly(A) tail attached to the free 3' end of the RNA by a template-dependent RNA polymerase after transcription, or a poly(A) tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

A polyA tail of approximately 120 A nucleotides has been shown to have a positive effect on the level of mRNA in transfected eukaryotic cells and on the level of protein translated from an open reading frame located upstream (5') of the polyA tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly-A tail can be of any length. In certain embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "essentially consisting" means that the majority of nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides in the poly A tail, i.e., 100% of the nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

In one embodiment, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (the coding strand) is referred to as a poly(A) cassette.

In one embodiment, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016/005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016/005324 A1 can be used in the present invention. Included are poly(A) cassettes having a length of, for example, 5 to 50 nucleotides, consisting essentially of dA nucleotides but interrupted by evenly distributed random sequences of four nucleotides (dA, dC, dG, dT), which, at the DNA level, exhibit consistent propagation of plasmid DNA in E. coli and, at the RNA level, are associated with beneficial properties related to supporting RNA stability and translation efficiency. As a result, in some embodiments, the poly-A tail contained in the RNA (particularly mRNA) molecules described herein consists essentially of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

In some embodiments, no nucleotides other than A nucleotides are adjacent to the poly-A tail at its 3' end, i.e., the poly-A tail is not masked or connected at its 3' end with nucleotides other than A.

In some embodiments, the poly-A tail can comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail can consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail can consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides. In some embodiments, the polyA tail comprises or consists of the nucleotide sequence of SEQ ID NO:3. In some embodiments, the polyA sequence has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identical to the nucleotide sequence of SEQ ID NO:3.

In some embodiments, an RNA (e.g., mRNA) used in the present invention includes a 5' UTR and/or a 3' UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5' UTR) and/or 3' (downstream) of an open reading frame (3' UTR). A 5' UTR, if present, is located at the 5' end, upstream of the start codon of a protein-coding region. A 5' UTR is downstream of the 5' cap (if present), e.g., immediately adjacent to the 5' cap. A 3' UTR, if present, is located at the 3' end, downstream of the stop codon of a protein-coding region, although the term "3' UTR" preferably does not include a poly(A) sequence. Thus, the 3' UTR is upstream of a polyA sequence (if present), e.g., immediately adjacent to the polyA sequence. Incorporation of a 3' UTR into the 3'-translated region of an RNA (preferably mRNA) molecule can result in enhanced translation efficiency. A synergistic effect can be achieved by incorporating two or more such 3' UTRs (preferably arranged in a head-to-tail orientation; see, e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)). The 3' UTR can be autologous or heterologous to the introduced RNA (preferably mRNA). In certain embodiments, the 3' UTR is derived from a globin gene or mRNA, such as alpha2-globin, alpha1-globin, or beta-globin, preferably beta-globin, more preferably human beta-globin. For example, RNA (preferably mRNA) can be modified by replacing or inserting one or more, preferably two copies of the existing 3' UTR from a globin gene, such as alpha2-globin, alpha1-globin, or beta-globin, preferably beta-globin, more preferably human beta-globin.

In one embodiment, an RNA (e.g., an mRNA) for use in the present invention comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:1.

In some embodiments, an RNA (e.g., an mRNA) for use in the present invention comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2.

The RNA (e.g., mRNA) described herein can have modified ribonucleotides to increase its stability and/or reduce its immunogenicity and/or reduce its cytotoxicity. For example, in some embodiments, uridines in the RNA (e.g., mRNA) described herein are replaced (partially or completely, preferably completely) with modified nucleosides. In some embodiments, the modified nucleoside is a modified uridine.

In one embodiment, the modified uridine replacing the uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

In some embodiments, modified nucleosides that replace (partially or completely, preferably completely) uridine in RNA (e.g., mRNA) include 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcm5U), and uridine 5-oxyacetic acid methyl ester (mcm5U). o5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carba 5-aminomethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1- Methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5 It may be one or more of carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely, replacing uridine) is referred to herein as "Ψ-modified," while the term "m1Ψ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely, replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely, replacing uridine). Such Ψ- or m1Ψ- or m5U-modified RNA typically exhibits reduced immunogenicity compared to unmodified forms and is therefore preferred in applications where induction of an immune response should be avoided or minimized. In one embodiment, the RNA (preferably mRNA) contains N(1)-methylpseudouridine, completely replacing uridine.

The codons of the RNA (preferably mRNA) described herein may be further optimized, for example, to increase the G/C content of the RNA and/or to replace codons that are rare in a cell (or subject) in which a peptide or protein of interest is to be expressed with codons that are frequent in the cell (or subject). In certain embodiments, the amino acid sequence encoded by the RNA described herein is encoded by a coding sequence that has been codon-optimized and/or has an increased G/C content compared to a wild-type coding sequence. This also includes embodiments in which one or more sequence regions of the coding sequence have been codon-optimized and/or have an increased G/C content compared to the corresponding sequence region of a wild-type coding sequence. In certain embodiments, the codon optimization and/or increased G/C content preferably does not alter the sequence of the encoded amino acid sequence.

The term "codon optimization" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism, preferably without altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, the coding region is preferably codon-optimized for optimal expression in a subject treated with the RNA (preferably mRNA) described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNAs in a cell. Thus, the sequence of the RNA (preferably mRNA) can be modified such that codons available for frequently occurring tRNAs are inserted in place of "rare codons."

In one embodiment, the guanosine/cytosine (G/C) content of the coding region of an RNA (preferably mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA (preferably mRNA) is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the discovery that the sequence of any RNA region to be translated is important for efficient translation of that RNA (preferably mRNA). Sequences with an increased G (guanosine)/C (cytosine) content are more stable than sequences with an increased A (adenosine)/U (uracil) content. Due to the fact that several codons encode the exact same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability can be determined (the so-called alternative codon frequency). Depending on the amino acid encoded by the RNA (preferably mRNA), there are various possibilities for modifying the RNA sequence compared to its wild-type sequence. In particular, codons containing A and/or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acids but that do not contain A and/or U nucleotides or that contain fewer A and/or U nucleotides.

In various embodiments, the G/C content of the coding region of the RNA (particularly mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55% or more compared to the G/C content of the coding region of the wild-type RNA.

The combination of the above modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, alteration of the 5'- and/or 3' UTR (e.g., incorporation of one or more 3' UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (Ψ) or N(1)-methylpseudouridine (m1Ψ) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic effect on RNA (preferably mRNA) stability and increases translation efficiency. Thus, in one embodiment,
The RNA (preferably mRNA) described herein, particularly the RNA (preferably mRNA) encoding the antigens or epitopes for inducing an immune response disclosed herein, can include any of the above-described modifications, namely, (i) incorporation of a 5' cap structure; (ii) incorporation of a polyA sequence, unmasking of a polyA sequence; (iii) modification of the 5'- and/or 3' UTR (e.g., incorporation of one or more 3'UTRs); (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., replacement of uridine with 5-methylcytidine and/or pseudouridine (Ψ) or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) for cytidine); and (v) combinations of at least two, at least three, at least four, or all five of the codon optimizations. In certain embodiments, the RNA (preferably mRNA) described herein includes a cap 1 or cap 2, preferably a cap 1 structure. In certain embodiments, the polyA sequence comprises at least 100 nucleotides. In some embodiments, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the 5' UTR comprises the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the 3' UTR comprises the nucleotide sequence of SEQ ID NO: 2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 2.

Certain aspects of the present invention involve targeted delivery of the RNA disclosed herein, preferably to: In certain embodiments, the present invention involves targeting of the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. Targeting of the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen, is particularly preferred if the administered RNA (preferably mRNA) is RNA (preferably mRNA) encoding an antigen or epitope for induction of an immune response. In certain embodiments, the target cell is a spleen cell. In certain embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. cells. In one embodiment, the target cells are dendritic cells in the spleen. The "lymphatic system" is the circulatory system, an important part of the immune system, and includes a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conductive network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including the lymph nodes and spleen, maintain mature naive lymphocytes and initiate adaptive immune responses. In one embodiment, the target cells are T cells.

Lipid-based RNA (e.g., mRNA) delivery systems have a unique preference for the liver. Liver accumulation is due to the discontinuous nature of the liver vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates). In certain embodiments, the target organ is the liver and the target tissue is hepatic tissue. Delivery to such target tissue is preferred, particularly if the presence of the mRNA or encoded peptide or protein in this organ or tissue is desired and/or expression of large amounts of the encoded peptide or protein is desired and/or systemic presence of the encoded peptide or protein, especially in substantial amounts, is desired or required.

In some embodiments, after administration of the RNA (particularly mRNA) compositions described herein, at least a portion of the RNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is RNA (preferably mRNA) that encodes a peptide or protein, and the RNA is translated in the target cell to produce the peptide or protein. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell of the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a lymph node cell. In some embodiments, the target cell is a lung cell. In some embodiments, the target cell is a skin cell. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cells are NK cells. In some embodiments, the target cells are monocytes. Thus, the nucleic acid (e.g., RNA) particle (e.g., RNA LNP) compositions described herein can be used to deliver nucleic acids (e.g., RNA, preferably mRNA) to such target cells. Accordingly, the present invention also relates to a method of delivering nucleic acids (e.g., RNA, preferably mRNA) to target cells in a subject, comprising administering to the subject a nucleic acid (e.g., RNA, preferably mRNA) composition described herein. In some embodiments, the RNA is delivered to the cytosol of the target cells. In some embodiments, the RNA is RNA (preferably mRNA) that encodes a peptide or protein, and the RNA is translated in the target cell to produce the peptide or protein.

Inhibitory RNA
In some embodiments of all aspects of the invention, the nucleic acid is an inhibitory RNA.

As used herein, the term "inhibitory RNA" refers to RNA that selectively hybridizes to and/or is specific for a target mRNA, thereby inhibiting (e.g., reducing) its transcription and/or translation. Inhibitory RNA includes RNA molecules having a sequence in an antisense orientation relative to a target mRNA. Suitable inhibitory oligonucleotides typically vary in length from 5 to several hundred nucleotides, more typically about 20-70 nucleotides in length or shorter, and even more typically about 10-30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozymes, iRNA, siRNA, and miRNA. In some embodiments of all aspects of the invention, the inhibitory RNA is siRNA.

As used herein, the term "antisense RNA" refers to RNA that, under physiological conditions, hybridizes with DNA containing a specific gene or with the mRNA of that gene, thereby inhibiting transcription of that gene and/or translation of that mRNA. An antisense transcript of a nucleic acid, or a portion thereof, forms a duplex with the naturally occurring mRNA, thus preventing accumulation or translation of the mRNA. Another possibility is the use of ribozymes to inactivate nucleic acids. Antisense RNA can hybridize with the N-terminus or 5' upstream site, such as the translation initiation site, transcription initiation site, or promoter site. In some embodiments, antisense RNA can hybridize with the 3' untranslated region or mRNA splicing site.

The size of the antisense RNA can vary from 15 nucleotides to 15,000, preferably 20-12,000, in particular 100-10,000, 150-8,000, 200-7,000, 250-6,000, 300-5,000 nucleotides, e.g., 15-2,000, 20-1,000, 25-800, 30-600, 35-500, 40-400, 45-300, 50-250, 55-200, 60-150 or 65-100 nucleotides. In some embodiments, the antisense RNA is at least 2,700 nucleotides in length (e.g., at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, or at least 5,000 nucleotides).

The stability of antisense RNA can be modified as needed. For example, antisense RNA can be modified with one or more modifications that have a stabilizing effect. Such modifications include modified phosphodiester linkages (e.g., methylphosphonate, phosphorothioate, phosphorodithioate, or phosphoramidate linkages in place of naturally occurring phosphodiester linkages) and 2'-substitutions (e.g., 2'-fluoro, 2'-O-alkyl (e.g., 2'-O-methyl, 2'-O-propyl, or 2'-O-pentyl) and 2'-O-allyl). For example, in some embodiments of antisense RNA, phosphorothioate linkages are partially substituted for phosphodiester linkages. Alternatively, or in addition, in some embodiments of antisense RNA, ribose moieties are partially substituted at the 2' position with O-alkyl (e.g., 2'-O-methyl).

Antisense RNA can be targeted to any stretch of approximately 19-25 contiguous nucleotides of any target mRNA sequence ("target sequence"). Generally, the target sequence of the target mRNA can be selected from a cDNA sequence corresponding to the target mRNA, preferably starting 50-100 nt downstream (i.e., in the 3' direction) from the start codon. However, the target sequence can also be located in the 5' or 3' untranslated region or in the region near the start codon.

Antisense RNA can be obtained using several techniques known to those skilled in the art. For example, antisense RNA can be chemically synthesized or recombinantly produced using methods known in the art. Preferably, antisense RNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter.

Selection of a suitable plasmid for expressing antisense RNA, methods for inserting a nucleic acid sequence into a plasmid for expression of antisense RNA, and IVT methods for in vitro transcription of the antisense RNA are within the skill of the art.

As used herein, "small interfering RNA" or "siRNA" refers to an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, capable of specifically binding to a portion of a target mRNA. This binding induces cleavage or degradation of that portion of the target mRNA, thereby inhibiting gene expression of the target mRNA. The 19-25 nucleotide range is the most preferred size for siRNA. While in principle, the sense and antisense strands of an siRNA can comprise two complementary, single-stranded RNA molecules, siRNA, according to the present invention comprises a single molecule in which the two complementary portions are base-paired and covalently linked by a single-stranded "hairpin" region. That is, the sense and antisense regions can be covalently linked via a linker molecule. The linker molecule can be a polynucleotide or non-nucleotide linker, but is preferably a polynucleotide linker. Without wishing to be bound by any theory, it is believed that the hairpin region of the siRNA molecule is cleaved by the "Dicer" protein (or its equivalent) within the cell, forming two individual base-paired RNA molecules of the siRNA.

The siRNA may also include a 3' overhang. As used herein, "3' overhang" refers to at least one unpaired nucleotide extending from the 3' end of an RNA strand. Thus, in certain embodiments, the siRNA includes at least one 3' overhang that is 1 to about 6 nucleotides (including ribonucleotides or deoxynucleotides) in length, preferably 1 to about 5 nucleotides in length, more preferably 1 to about 4 nucleotides in length, and particularly preferably about 2 to about 4 nucleotides in length. In embodiments in which both strands of the siRNA molecule (i.e., after the siRNA molecule is cleaved by Dicer protein in a cell) include a 3' overhang, the length of the overhangs can be the same or different for each strand. In certain preferred embodiments, the 3' overhangs are present on both strands of the siRNA and are 2 nucleotides in length. For example, each strand of the siRNA can include a 3' overhang of dideoxythymidylic acid ("TT") or diuridylic acid ("uu").

To enhance the stability of siRNA, the 3' overhangs can also be stabilized against degradation. In some embodiments, the overhangs are stabilized by the inclusion of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of modified analogs of pyrimidine nucleotides, e.g., substitution of uridine nucleotides in the 3' overhang with 2'-deoxythymidine, is tolerated and does not affect the efficiency of RNAi degradation. In particular, the absence of a 2'-hydroxyl in 2'-deoxythymidine significantly enhances the nuclease resistance of the 3' overhang in tissue culture medium.

As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide identified herein. In certain embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (particularly, e.g., increased expression compared to expression in healthy subjects) is associated with disease. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (particularly, e.g., increased expression compared to expression in healthy subjects) is associated with cancer.

According to the present invention, siRNA can target any stretch of about 19 to 25 contiguous nucleotides of any target mRNA sequence ("target sequence"). Techniques for selecting target sequences for siRNA are set forth, for example, in Tuschl T. et al., "The siRNA User Guide," revised Oct. 11, 2002, the entire disclosure of which is incorporated herein by reference. "The siRNA User Guide" is available on the Internet at the website maintained by Dr. Thomas Tuschl, Laboratory of RNA Molecular Biology, Rockefeller University, New York, USA, which can be found by accessing the Rockefeller University website and searching for the keyword "siRNA." Further guidance on target sequence selection and/or siRNA design can be found on the homepage of Protocol Online (www.protocol-online.com) using the keyword "siRNA." Thus, in certain embodiments, the sense strand of the siRNA used in the present invention comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides of the target mRNA.

Generally, the target sequence of the target mRNA can be selected from a cDNA sequence corresponding to the target mRNA, preferably starting 50-100 nt downstream (i.e., in the 3' direction) from the start codon. However, the target sequence can also be located in the 5' or 3' untranslated region or in the region near the start codon.

siRNA can be obtained using several techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. Application No. 2002/0086356 to Tuschl et al., the entire disclosure of which is incorporated herein by reference. siRNA can also be expressed from Pol III expression vectors without altering the targeting site, since RNA expression from Pol III promoters is believed to be efficient only when the first transcribed nucleotide is a purine.

Preferably, siRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter. Suitable promoters for transcribing siRNA for use in the present invention from a plasmid include, for example, the U6 or H1 RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill of the art.

Selection of a suitable plasmid for transcribing siRNA, methods for inserting a nucleic acid sequence for expression of siRNA into the plasmid, and IVT methods for in vitro transcription of the siRNA are within the skill of the art.

As used herein, the term "miRNA" (microRNA) refers to a non-coding RNA that is 21-25 (e.g., 21-23, preferably 22) nucleotides in length and induces degradation of target mRNA or blocks translation. miRNAs are typically found in plants, animals, and certain viruses, where they are encoded by eukaryotic nuclear DNA and viral DNA (in viruses whose genomes are DNA-based), respectively. miRNAs are post-transcriptional regulators that typically bind to complementary sequences in target messenger RNA transcripts (mRNAs), resulting in translational repression or target degradation and gene silencing.

miRNA can be obtained using several techniques known to those skilled in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., using a commercially available kit such as the miRNA cDNA synthesis kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from a recombinant circular or linear DNA plasmid using any suitable promoter.

"Encoding" a pharmaceutically active peptide or polypeptide refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or RNA (preferably mRNA), to serve as a template in biological processes for the synthesis of other polymers and macromolecules having a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids and the biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the RNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

In certain embodiments, the RNA (preferably mRNA) described herein comprises a nucleic acid sequence (e.g., an ORF) that encodes one or more polypeptides, e.g., peptides or proteins, preferably pharmaceutically active peptides or proteins.

In certain embodiments, the RNA (preferably mRNA) described herein comprises a nucleic acid sequence (e.g., an ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein, and is particularly capable of expressing the peptide or protein when introduced into a cell or a subject. Thus, in certain embodiments, the RNA (preferably mRNA) described herein comprises a coding region (ORF) encoding a peptide or protein, preferably a pharmaceutically active peptide or protein. In this regard, an "open reading frame" or "ORF" is a contiguous stretch of codons beginning with an initiation codon and ending with a termination codon. RNA (preferably mRNA) encoding such a pharmaceutically active peptide or protein is also referred to herein as "pharmaceutically active RNA" (or "pharmaceutically active mRNA"). In certain embodiments, the RNA (preferably mRNA) described herein comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides.

The term "pharmaceutically active RNA" also encompasses RNA that is itself pharmaceutically active; thus, pharmaceutically active RNA can alternatively be antisense RNA, one or more strands of microRNA (miRNA), siRNA, RNA interference (RNAi), short hairpin RNA (shRNA), or non-coding RNA such as a precursor of siRNA or microRNA-like RNA that targets a target transcript of interest, e.g., a transcript of an endogenous disease-associated transcript.

According to the present invention, the term "pharmaceutically active peptide or protein" refers to a peptide or protein that can be used to treat an individual, where expression of the peptide or protein is beneficial, for example, in alleviating the symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and can be used to improve, alleviate, mitigate, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. In some embodiments, a pharmaceutically active peptide or protein, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or disease state. A pharmaceutically active peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or disorder or reduce the severity of such a disease or disorder. The term "pharmaceutically active peptide or protein" can include the entire protein or polypeptide, as well as pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of the peptide or protein. The terms "pharmaceutically active peptide or protein" and "therapeutic peptide or protein" are used interchangeably herein.

Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, immune stimulators, e.g., cytokines, hormones, adhesion molecules, immunoglobulins, immunologically active compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome engineering proteins, and blood proteins. In some embodiments, pharmaceutically active peptides and polypeptides include substituted proteins.

An "immunostimulator" is any substance that stimulates the immune system by increasing the activation or activity of any of the components of the immune system, particularly immune effector cells. Immune stimulators can be pro-inflammatory (e.g., when treating infection or cancer) or anti-inflammatory (e.g., when treating autoimmune diseases).

In one embodiment, the immune stimulatory factor is a cytokine or a variant thereof. Examples of cytokines include interferons, such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins, such as IL2, IL7, IL12, IL15, and IL23, colony-stimulating factors, such as M-CSF and GM-CSF, and tumor necrosis factor. According to another embodiment, the immune stimulatory factor comprises an adjuvant-type immune stimulator, such as an APC toll-like receptor agonist or a costimulatory/cell adhesion membrane protein. Examples of toll-like receptor agonists include costimulatory/adhesion proteins, such as CD80, CD86, and ICAM-1.

The term "cytokine" refers to a protein having a molecular weight of approximately 5-60 kDa (e.g., approximately 5-20 kDa) and participating in cell signaling (e.g., paracrine, endocrine, and/or autocrine signaling). In particular, when released, cytokines affect the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present invention, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that modify and regulate gene transcription within cells by binding to interferon receptors on the surface of regulatory cells, thereby inhibiting viral replication within the cells. Interferons can be grouped into two types: IFN-gamma is the only type II interferon; all others are type I interferons. Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte-colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (IFNγ), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12 (IL-12), and interleukin-21 (IL-21).

According to the present invention, the cytokine may be a naturally occurring cytokine or a functional fragment or variant thereof. The cytokine may be a human cytokine and may be derived from any vertebrate, particularly any mammal. One particularly preferred cytokine is interferon-α.

An immune stimulatory factor may be provided to a subject by administering to the subject a nucleic acid (e.g., DNA or RNA) encoding the immune stimulatory factor in a formulation for preferential delivery of the nucleic acid to the liver or liver tissue. Delivery of nucleic acid (e.g., DNA or RNA) to such a target organ or tissue is preferred, particularly if expression of large amounts of the immune stimulatory factor is desired and/or if systemic presence of the immune stimulatory factor, especially in substantial amounts, is desired or required.

Nucleic acid (e.g., DNA or RNA) delivery systems have a unique preference for the liver. This is relevant for lipid-based particles, cationic and neutral nanoparticles, and particularly lipid nanoparticles.

Examples of suitable immune stimulators for targeting the liver include cytokines involved in T-cell proliferation and/or maintenance. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

In certain embodiments, nucleic acids (e.g., DNA or RNA) encoding an immunostimulatory agent may be administered in a formulation for preferential delivery of the nucleic acid (e.g., DNA or RNA) to the lymphatic system, particularly to secondary lymphoid organs, more particularly to the spleen. Delivery of the immunostimulatory agent to such a target tissue may be preferred, particularly when the presence of the immunostimulatory agent in this organ or tissue is desired (e.g., for induction of an immune response, particularly when the immunostimulatory agent, such as a cytokine, is required during T cell priming or for resident immune cell activation), but when a systemic presence of the immunostimulatory agent, particularly in significant amounts, is undesirable (e.g., because the immunostimulatory agent has systemic toxicity).

Examples of suitable immune stimulators include cytokines involved in T cell priming. Examples of suitable cytokines include IL12, IL15, IFN-α or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and even tumor cells. In a typical scenario, virus-infected cells release interferons to mount the antiviral defenses of nearby cells.

Based on the type of receptor through which they signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

All type I interferons bind to a specific cell surface receptor complex known as the IFN-α/β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

Type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, leading to the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs). They are primarily involved in innate immunity against viral infections. The genes responsible for their synthesis are divided into 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

IFNβ protein is produced in large quantities by fibroblasts. It has antiviral activity, primarily involved in the innate immune response. Two types of IFNβ have been described: IFNβ1 and IFNβ3. Natural and recombinant forms of IFNβ1 have antiviral, antibacterial, and anticancer properties.

Type II interferon (IFNγ in humans), also known as immune interferon, is activated by IL-12. Additionally, type II interferon is released by cytotoxic T cells and T helper cells.

Type III interferons signal through a receptor complex consisting of IL10R2 (also known as CRF2-4) and IFNLR1 (also known as CRF2-12). Although discovered more recently than type I and type II IFNs, recent information indicates the importance of type III IFNs in certain types of viral and fungal infections.

In general, type I and type II interferons are responsible for regulating and activating the immune response.

According to the present invention, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

According to the present invention, the interferon may be a naturally occurring interferon or a functional fragment or variant thereof. The interferon may be a human interferon and may be derived from any vertebrate, particularly any mammal.

Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on characteristic structural features. However, amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

According to the present invention, the interleukin may be a naturally occurring interleukin or a functional fragment or variant thereof. The interleukin may be a human interleukin and may be derived from any vertebrate, particularly any mammal.

The immunostimulatory polypeptides described herein can be produced as fusion or chimeric polypeptides comprising an immunostimulatory portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory portion). The immunostimulatory portion can be fused to an extended PK group that extends circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that extend the circulating half-life of immunostimulators, such as cytokines or variants thereof, are also applicable to the present invention. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

As used herein, the term "PK" is an acronym for "pharmacokinetic" and encompasses the properties of a compound, including, by way of example, absorption, distribution, metabolism, and excretion by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, extends the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Publications 2005/0287153 and 2007/0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, incorporated herein by reference in its entirety. As used herein, an "extended PK" immunostimulator refers to an immunostimulatory factor moiety combined with an extended PK group. In some embodiments, the extended PK immunostimulator is a fusion protein in which the immunostimulatory factor moiety is linked or fused to the extended PK group.

In some embodiments, the serum half-life of the extended PK immunostimulator is increased compared to the immunostimulator alone (i.e., the immunostimulator not fused to an extended PK group). In some embodiments, the serum half-life of the extended PK immunostimulator is at least 20%, at least 40%, at least 60%, at least 80%, at least 100%, at least 120%, at least 150%, at least 180%, at least 200%, at least 400%, at least 600%, at least 800%, or at least 1000% longer than the serum half-life of the immunostimulator alone. In some embodiments, the serum half-life of the extended PK immunostimulator is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of the immunostimulator alone. In some embodiments, the serum half-life of the extended PK immunostimulator is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

As used herein, "half-life" refers to the time it takes for the serum or plasma concentration of a compound, such as a peptide or polypeptide, to decrease by 50% in vivo, e.g., due to degradation and/or clearance or sequestration by natural mechanisms. Extended PK immunostimulators suitable for use herein are stabilized in vivo, and their half-life is extended, e.g., by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and/or clearance or sequestration. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and generally involve, for example, administering an appropriate dose of an amino acid sequence or compound to a suitable subject; obtaining blood or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating the time it takes for the level or concentration of the amino acid sequence or compound to decrease by 50% compared to the initial level at the time of administration, based on a plot of the data thus obtained. Further details are provided in standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

In some embodiments, the extended PK group comprises serum albumin or a fragment thereof or a variant of serum albumin or a fragment thereof (all of which are encompassed by the term "albumin" for purposes of the present invention). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

As used herein, the term "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a therapeutic protein, particularly a protein such as an immunostimulator. Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is joined in-frame with a polynucleotide encoding albumin. Once part of an albumin fusion protein, the therapeutic protein and albumin can each be referred to as a "portion," "region," or "moiety" of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In highly preferred embodiments, the albumin fusion protein comprises at least one molecule of a therapeutic protein (including, but not limited to, the mature form of a therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In certain embodiments, the albumin fusion protein is processed by host cells, such as cells of a target organ for the administered RNA, e.g., liver cells, and secreted into the circulation. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA includes, but is not limited to, signal peptide cleavage; disulfide bond formation; proper folding; carbohydrate addition and processing (e.g., N- and O-linked glycosylation); specific proteolytic cleavage; and/or assembly into multimers. The albumin fusion protein is preferably encoded by the RNA in an unprocessed form, particularly with a signal peptide at its N-terminus, and, following secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In a most preferred embodiment, "processed form of albumin fusion protein" refers to an albumin fusion protein product that has been subjected to N-terminal signal peptide cleavage, also referred to herein as "mature albumin fusion protein."

In certain embodiments, the albumin fusion protein comprises a therapeutic protein that has increased plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period between when the therapeutic protein is administered in vivo and transported into the bloodstream, and when the therapeutic protein is degraded and removed from the bloodstream to organs such as the kidneys or liver, which ultimately eliminate the therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

As used herein, "albumin" collectively refers to the albumin protein or amino acid sequence, or to albumin fragments or variants having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin from other vertebrates, or a fragment or variant of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include chicken and salmon. The albumin portion of an albumin fusion protein can be derived from a different animal than the therapeutic protein portion.

In one embodiment, the albumin is human serum albumin (HSA) or a fragment or variant thereof, such as those described in US Pat. No. 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broad and include human serum albumin (and fragments and variants thereof) and albumins (and fragments and variants thereof) from other species.

As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability of the unfused state.

The albumin portion of the albumin fusion protein may have the full-length albumin sequence or may include one or more fragments thereof that are capable of stabilizing or extending therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may include about 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may include some or all of a specific domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

According to the present invention, the albumin may be naturally occurring albumin or a fragment or variant thereof. The albumin may be human albumin and may be derived from any vertebrate, particularly any mammal.

In one embodiment, the albumin fusion protein comprises albumin as the N-terminal portion and a Therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion and a Therapeutic protein as the N-terminal portion may also be used. In another embodiment, the albumin fusion protein has Therapeutic proteins fused to both the N- and C-termini of albumin. In a preferred embodiment, the Therapeutic proteins fused to the N- and C-termini are the same Therapeutic protein. In another preferred embodiment, the Therapeutic proteins fused to the N- and C-termini are different Therapeutic proteins. In one embodiment, the different Therapeutic proteins are both cytokines.

In some embodiments, a therapeutic protein can be linked to albumin via a peptide linker. A peptide linker between fusion moieties can increase the physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety for binding to its cognate receptor, for example. The peptide linker can be composed of amino acids to be flexible or more rigid. The linker sequence can be protease or chemically cleavable.

As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, where the Fc domain does not include an Fv domain. In certain embodiments, an Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion) fused to a CH3 domain (or portion). In some embodiments, the Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In some embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain herein generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and/or CH3 domains, as well as fragments of such peptides, e.g., comprising only the hinge, CH2, and CH3 domains. The Fc domain can be derived from any species and/or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass native Fc and Fc variant molecules. As provided herein, those skilled in the art will understand that an Fc domain can be modified to vary in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the effector function (e.g., FcγR binding) of the Fc domain is reduced.

The Fc domains of the polypeptides described herein can be derived from different immunoglobulin molecules. For example, the Fc domain of the polypeptide can include a CH2 and/or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain can include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domain can include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.

In one embodiment, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are encompassed by the term "Fc domain" for purposes of the present invention). An Fc domain does not contain a variable region that binds to an antigen. Fc domains suitable for use in the present invention can be obtained from several different sources. In one embodiment, the Fc domain is derived from a human immunoglobulin. In one embodiment, the Fc domain is derived from a human IgG1 constant region. However, the Fc domain can also be derived from immunoglobulins of other mammalian species, including, for example, rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

Furthermore, the Fc domain (or fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

A variety of Fc domain gene sequences (e.g., murine and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains can be selected, including Fc domain sequences that lack specific effector functions and/or have specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and appropriate Fc domain sequences (e.g., hinge, CH2, and/or _CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art-recognized techniques.

In some embodiments, the extended PK group comprises a serum albumin binding protein, such as those described in US 2005/0287153, US 2007/0003549, US 2007/0178082, US 2007/0269422, US 2010/0113339, WO 2009/083804, and WO 2009/133208, which are incorporated herein by reference in their entireties. In some embodiments, the extended PK group is transferrin, as disclosed in US 7,176,278 and US 8,158,579, which are incorporated herein by reference in their entireties. In some embodiments, the extended PK group is a serum immunoglobulin-binding protein, such as those disclosed in US 2007/0178082, US 2014/0220017, and US 2017/0145062, which are incorporated herein by reference in their entireties. In some embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US 2012/0094909, which are incorporated herein by reference in their entireties. Methods for producing fibronectin-based scaffold domain proteins are disclosed in US 2012/0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

In some embodiments, an extended PK immunostimulatory agent suitable for use according to the present invention may employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., an extended PK portion and an immunostimulatory agent portion) in the linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to connect an immunostimulatory agent portion to an HSA domain.

Linkers suitable for fusing extended PK groups to, for example, immunostimulatory factors are well known in the art. Exemplary linkers include glycine-serine-polypeptide linkers, glycine-proline-polypeptide linkers, and proline-alanine polypeptide linkers. In some embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

In some embodiments, the pharmaceutically active peptide or protein comprises a replacement protein. In these embodiments, the present invention provides methods of treating a subject having a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject an RNA described herein that encodes the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) into a subject having a deficiency of such protein. The term also refers to the introduction of a protein into a subject who otherwise requires or would benefit from the provision of protein, e.g., a subject with a protein deficiency. The term "disorder characterized by protein deficiency" refers to any disorder exhibiting pathology due to the absence or insufficient amount of protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in a biologically inactive protein product. Protein deficiency can be associated with infection, immunosuppression, organ failure, glandular disease, radiation sickness, nutritional deficiency, poisoning, or other environmental or external insults.

The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction typically involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) signal relay and amplification; and (vi) degradation of the hormone. Hormones differ from cytokines in that (1) hormones typically act at relatively low concentrations and (2) are generally produced by cells of specific species. In some embodiments, a "hormone" is a peptide or protein hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (e.g., human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

The term "adhesion molecule" refers to proteins located on the cell surface that are involved in the binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and are divided into calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules are integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cell signaling pathways, for example, pathways of transmembrane protein kinases such as _receptor tyrosine kinases (RTKs). Such regulation can lead to cell growth _{, division} , _survival , differentiation, or _apoptosis . Specific examples of integrins _{include α1β1} , _α2β1 , _α3β1 , _α4β1 , _{α5β1 , α6β1} , _α7β1 , _{αLβ2 , αMβ2 , αIIbβ3 , αVβ1 , αVβ3 , αVβ5 , αVβ6 , αVβ8 , and α6β4 .}

The terms "immunoglobulin" or "immunoglobulin superfamily" refer to molecules involved in cell recognition, binding, and/or adhesion processes. Molecules belonging to this superfamily share the property of containing regions known as immunoglobulin domains or folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD3-gamma, CD3-delta, CD3-epsilon, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and others.

The term "immunologically active compound" relates to any compound that modifies the immune response, preferably by altering humoral immunity by inducing and/or inhibiting immune cell maturation, inducing and/or inhibiting cytokine biosynthesis, and/or stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can downregulate other aspects of the immune response, shifting the immune response away from a TH2 immune response, which is useful, for example, in the treatment of a wide range of TH2-mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants. Specific examples of immunologically active compounds are interleukins, colony stimulating factors (CSFs), granulocyte colony stimulating factors (G-CSFs), granulocyte-macrophage colony stimulating factors (GM-CSFs), erythropoietin, tumor necrosis factors (TNFs), interferons, integrins, addressins, selectins, homing receptors, T-cell receptors, chimeric antigen receptors (CARs), immunoglobulins and antigens, in particular tumor-associated antigens, pathogen-associated antigens (e.g. bacterial, parasitic or viral antigens), allergens, autoantigens, hormones, and the like. hormones (insulin, thyroid hormones, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthesis or degradation, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases) , dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate or guanylate cyclase, neuraminidase, lysosomal enzymes, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, etc.), transcription and translation factors, tumor growth suppressor proteins (e.g., proteins that inhibit angiogenesis), structural proteins (e.g., collagen, fibroin, fibrinogen, elastin, tubulin, etc.), and the like. Examples of immunologically active compounds include antibodies against erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified factor VIII, anticoagulants, and the like. Preferred immunologically active compounds are vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response.

In certain embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein comprise a nucleic acid sequence encoding a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject. A "peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject" is also referred to herein as a "vaccine antigen," "peptide and protein antigen," or simply "antigen."

In one embodiment, the RNA (particularly, mRNA) encoding the vaccine antigen is a single-stranded, 5'-capped mRNA that is translated into the respective protein upon entry into the cells of the subject receiving the RNA, e.g., antigen-presenting cells (APCs). Preferably, the RNA (i) contains structural elements optimized for maximum RNA efficacy with respect to stability and translation efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified to optimize RNA efficacy (e.g., increased translation efficiency, reduced immunogenicity, and/or reduced cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (particularly cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine); and/or by codon optimization), or (iii) both (i) and (ii).

In one embodiment, beta-S-ARCA (D1) is utilized as a specific capping structure at the 5' end of the RNA. In one embodiment, the 5' UTR comprises the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:1. In one embodiment, the 3' UTR comprises the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2. In one embodiment, the poly(A) sequence is 110 nucleotides in length and consists of 30 adenosine residues, followed by a 10-nucleotide linker sequence and a stretch of an additional 70 adenosine residues. This poly(A) sequence is designed for enhanced RNA stability and translation efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:3 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:3. In some embodiments, the RNA comprises modified nucleosides in place of uridines. In some embodiments, the modified nucleosides that replace uridines (partially or completely, preferably completely) are selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine. In some embodiments, the RNA encoding the vaccine antigen has a coding sequence that is (a) codon-optimized, (b) has an increased G/C content compared to the wild-type coding sequence, or (c) both (a) and (b).

In some embodiments, nucleic acid (e.g., DNA or RNA) encoding a vaccine antigen is expressed in cells of a subject to provide the vaccine antigen. In some embodiments, expression of the vaccine antigen is at the cell surface. In some embodiments, the vaccine antigen is presented in the context of MHC. In some embodiments, nucleic acid (e.g., DNA or RNA) encoding a vaccine antigen is transiently expressed in cells of a subject. In some embodiments, nucleic acid (e.g., DNA or RNA) encoding a vaccine antigen is administered systemically. In some embodiments, systemic administration of nucleic acid (e.g., DNA or RNA) encoding a vaccine antigen results in expression of the nucleic acid encoding the vaccine antigen in the spleen. In some embodiments, systemic administration of nucleic acid encoding a vaccine antigen results in expression of the nucleic acid encoding the vaccine antigen in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, systemic administration of nucleic acid encoding a vaccine antigen results in no or essentially no expression of the nucleic acid encoding the vaccine antigen in the lung and/or liver. In some embodiments, systemic administration of nucleic acid encoding a vaccine antigen results in expression of the nucleic acid encoding the vaccine antigen in the spleen that is at least five times the amount of expression in the lung.

A vaccine antigen comprises an epitope for inducing an immune response against the antigen in a subject. Thus, a vaccine antigen comprises an antigenic sequence for inducing an immune response against the antigen in a subject. Such an antigenic sequence may correspond to a target antigen or disease-associated antigen, e.g., a protein of an infectious agent (e.g., a viral or bacterial antigen) or a tumor antigen, or an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-associated antigen or an immunogenic variant thereof. Thus, the antigenic sequence may comprise at least an epitope of the target antigen or disease-associated antigen or an immunogenic variant thereof.

Antigenic sequences, e.g., epitopes, suitable for use in accordance with the present invention may typically be derived from target antigens, i.e., antigens against which an immune response is to be generated. For example, the antigenic sequence contained in a vaccine antigen may be the target antigen or a fragment or variant of the target antigen.

The antigenic sequence or its progressive product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR or CAR, carried by an immune effector cell. In one embodiment, the antigenic sequence is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cell, or a variant of the antigenic sequence or fragment.

The vaccine antigens provided to a subject by the present invention through administration of nucleic acid (e.g., DNA or RNA) encoding the vaccine antigen preferably result in the induction of an immune response, e.g., stimulation, priming, and/or expansion of immune effector cells, in the subject to which the vaccine antigen is provided. The immune response, e.g., stimulated, primed, and/or expanded immune effector cells, is preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and/or organs, i.e., a disease-associated antigen. Thus, the vaccine antigen may comprise a disease-associated antigen or a fragment or variant thereof. In certain embodiments, such a fragment or variant is immunologically equivalent to the disease-associated antigen.

In the present invention, the term "antigen fragment" or "antigen variant" refers to an agent that results in the induction of an immune response, e.g., stimulation, priming, and/or expansion of immune effector cells, where the immune response, e.g., stimulation, priming, and/or expansion of immune effector cells, targets an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, tissues, and/or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, may correspond to or comprise a fragment of a disease-associated antigen, or may correspond to or comprise an antigen homologous to the disease-associated antigen or its fragment. If a vaccine antigen comprises a fragment or amino acid sequence of a disease-associated antigen that is homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen targeted by the antigen receptor of an immune effector cell or a sequence homologous to an epitope of the disease-associated antigen. Thus, according to the present invention, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. According to the present invention, "immunogenic antigen fragment" preferably relates to a fragment of an antigen capable of inducing an immune response, e.g., stimulating, priming, and/or expanding immune effector cells carrying an antigen receptor bound to the antigen or cells expressing the antigen. Preferably, the vaccine antigen (similar to a disease-associated antigen) provides relevant epitopes upon binding by antigen receptors present on immune effector cells. In one embodiment, the vaccine antigen or a fragment thereof (similar to a disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell (optionally in the context of an MHC), to provide relevant epitopes for binding by immune effector cells. The vaccine antigen may be a recombinant antigen.

In some embodiments of all aspects of the invention, nucleic acid (e.g., DNA or RNA) encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, which binding results in stimulation, priming, and/or expansion of the immune effector cells.

In some embodiments, the antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. The antigen or its processed product, such as a T-cell epitope, is, in some embodiments, bound by an antigen receptor. Thus, the antigen or its processed product can specifically react with an immune effector cell, such as a T-lymphocyte (T-cell).

The term "autoantigen" or "self-antigen" refers to an antigen that originates within a subject's body (i.e., an autoantigen can also be referred to as a "self-antigen") and produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen can be the cause of an "autoimmune disease."

In some embodiments, the antigen is expressed on the surface of a diseased cell (e.g., a tumor cell or an infected cell). In some embodiments, the antigen receptor is a CAR that binds to an extracellular domain or an epitope in the extracellular domain of the antigen. In some embodiments, the CAR binds to an epitope of a native antigen present on the surface of a living cell. In some embodiments, binding of the CAR, when expressed by a T cell and/or presented on a T cell in response to an antigen presented on a cell, such as an antigen-presenting cell, results in stimulation, priming, and/or expansion of the T cell. In some embodiments, binding of the CAR, when expressed by a T cell and/or presented on a T cell in response to an antigen present on a diseased cell, results in cytolysis and/or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors, e.g., perforin and granzymes.

According to some embodiments, the amino acid sequence that enhances antigen processing and/or presentation is fused to an antigenic peptide or polypeptide (antigenic sequence) directly or via a linker. Thus, in some embodiments, the nucleic acids (e.g., DNA or RNA) described herein comprise at least one coding region that encodes an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and/or presentation.

In some embodiments, antigens for vaccination may be administered in the form of nucleic acids (e.g., DNA or RNA) encoding the antigen, including naturally occurring antigens or fragments thereof, such as epitopes thereof.

Such antigen processing and/or presentation-enhancing amino acid sequences are preferably, but not limited to, located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the C-terminus of the immune tolerance-breaking amino acid sequence). The antigen processing and/or presentation-enhancing amino acid sequences defined herein preferably improve antigen processing and presentation. In certain embodiments, the antigen processing and/or presentation-enhancing amino acid sequences defined herein include, but are not limited to, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), particularly a sequence comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof.

In some embodiments, a secretory sequence, e.g., a sequence comprising the amino acid sequence of SEQ ID NO: 4, can be fused to the N-terminus of an antigenic peptide or polypeptide.

In one embodiment, the amino acid sequence that enhances antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO:5, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5, or a functional fragment of the amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:5. In one embodiment, the amino acid sequence that enhances antigen processing and/or presentation comprises the amino acid sequence of SEQ ID NO:5.

Thus, in some embodiments, a nucleic acid (e.g., DNA or RNA) described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and/or presentation, wherein the amino acid sequence that enhances antigen processing and/or presentation is preferably fused to the antigenic peptide or polypeptide, as described herein, more preferably to the C-terminus of the antigenic peptide or polypeptide.

Additionally, a secretory sequence, for example, a sequence comprising the amino acid sequence of SEQ ID NO: 4, can be fused to the N-terminus of the antigenic peptide or polypeptide.

An amino acid sequence derived from tetanus toxoid from Clostridium tetani can be used to overcome self-tolerance mechanisms to efficiently mount an immune response to self-antigens through T cell help during priming.

Tetanus toxoid heavy chains are known to contain epitopes that bind promiscuously to MHC class II alleles and can induce CD4 ⁺ memory T cells in nearly all tetanus-vaccinated individuals. Furthermore, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens is known to improve immune stimulation compared to tumor-associated antigens alone by providing CD4 ⁺ -mediated T cell help during priming. To reduce the risk of CD8 ⁺ T cell stimulation with tetanus sequences competing with the intended induction of tumor antigen-specific T cell responses, we avoided using the entire fragment C of tetanus toxoid, which is known to contain CD8 ⁺ T cell epitopes. Alternatively, two peptide sequences containing promiscuous helper epitopes were selected to ensure binding to as many MHC class II alleles as possible. Based on data from ex vivo studies, the well-known epitopes p2 (QYIKANSKFIGITEL; TT _830-844 ; SEQ ID NO: 9) and p16 (MTNSVDDALINSTKIYSYFPSVISKVNQGAQG; TT _578-609 ; SEQ ID NO: 10) were selected. The p2 epitope has already been used as a peptide vaccination in clinical trials to boost anti-melanoma activity.

Preclinical data indicate that RNA encoding both tumor antigens and promiscuous tetanus toxoid sequences enhances CD8 ⁺ T cell responses to tumor antigens and ameliorates tolerance breakdown. Immune monitoring data from patients vaccinated with vaccines containing sequences fused in-frame with tumor antigen-specific sequences confirm that the selected tetanus sequences were able to induce tetanus-specific T cell responses in nearly all patients.

In one embodiment, the tolerance-disrupting amino acid sequence is fused to the antigenic peptide or polypeptide either directly or via a linker, e.g., a linker having the amino acid sequence of SEQ ID NO: 7.

Such an amino acid sequence that disrupts immune tolerance is preferably, but is not limited to, located at the C-terminus of the antigenic peptide or polypeptide (and optionally at the N-terminus of the amino acid sequence that enhances antigen processing and/or presentation, where the amino acid sequence that disrupts immune tolerance and the amino acid sequence that enhances antigen processing and/or presentation can be fused directly or via a linker, e.g., a linker having the amino acid sequence of SEQ ID NO: 8). The amino acid sequence that disrupts immune tolerance defined herein preferably improves T cell responses. In one embodiment, the amino acid sequence that disrupts immune tolerance defined herein includes, but is not limited to, a sequence derived from tetanus toxoid-derived helper sequences p2 and p16 (P2P16), particularly a sequence comprising the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

In one embodiment, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:6, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6, or a functional fragment of the amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:6. In one embodiment, the amino acid sequence that disrupts immune tolerance comprises the amino acid sequence of SEQ ID NO:6.

Next, embodiments of the vaccine RNA will be described, wherein certain terms used when describing elements thereof have the following meanings:
Cap: A 5' cap structure selected from the group consisting of m ₂ ^7,2'O G(5')pppSp(5')G (especially its D1 diastereomer), m ₂ ^7,3'O G(5')ppp(5')G and m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG.

hAg-Kozak: 5' UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency.

sec/MITD: A fusion protein tag derived from sequences encoding the human MHC class I complex (HLA-B51, haplotype A2, B27/B51, Cw2/Cw3), which has been shown to improve antigen processing and presentation. Sec corresponds to a 78-bp fragment encoding a secretory signal peptide that guides translocation of the nascent polypeptide chain to the endoplasmic reticulum. MITD corresponds to the transmembrane and cytoplasmic domain of the MHC class I molecule, also known as the MHC class I transport domain.

Antigen: The sequence encoding each vaccine antigen/epitope.

Glycine-serine linker (GS): A sequence encoding a short peptide linker consisting primarily of the amino acids glycine (G) and serine (S), commonly used in fusion proteins.

P2P16: A sequence encoding a tetanus toxoid-derived helper epitope for breaking immune tolerance.

FI element: The 3' UTR is a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (termed FTP) and the mitochondrially encoded 12S ribosomal RNA (termed I). These were identified through an ex vivo selection process for sequences that confer enhanced RNA stability and total protein expression.

A30L70: A poly(A) tail up to 110 nucleotides long, consisting of 30 adenosine residues followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues, designed to enhance RNA stability and translation efficiency in dendritic cells.

In one embodiment, the vaccine RNA described herein has the following structure:
Cap-hAg-Kozak-sec-GS(1) antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70
beta-S-ARCA(D1)-hAg-Kozak-sec-GS(1) antigen-GS(2)-P2P16-GS(3)-MITD-FI-A30L70
It has one of the following.

In one embodiment, the vaccine antigen described herein has the structure:
sec-GS(1) antigen-GS(2)-P2P16-GS(3)-MITD
It has.

In one embodiment, hAg-Kozak comprises the nucleotide sequence of SEQ ID NO:1. In one embodiment, sec comprises the amino acid sequence of SEQ ID NO:4. In one embodiment, P2P16 comprises the amino acid sequence of SEQ ID NO:6. In one embodiment, MITD comprises the amino acid sequence of SEQ ID NO:5. In one embodiment, GS(1) comprises the amino acid sequence of SEQ ID NO:7. In one embodiment, GS(2) comprises the amino acid sequence of SEQ ID NO:8. In one embodiment, GS(3) comprises the amino acid sequence of SEQ ID NO:8. In one embodiment, FI comprises the nucleotide sequence of SEQ ID NO:2. In one embodiment, A30L70 comprises the nucleotide sequence of SEQ ID NO:3.

In one embodiment, the sequence encoding the vaccine antigen/epitope includes modified nucleosides replacing uridines (partially or completely, preferably completely), where the modified nucleosides are selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine.

In one embodiment, the sequence encoding the vaccine antigen/epitope is codon-optimized.

In some embodiments, the G/C content of the sequence encoding the vaccine antigen/epitope is increased compared to the wild-type coding sequence.

The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. If a T cell interacts with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon-gamma. Examples of non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

The term "dendritic cell" (DC) refers to a subtype of phagocyte belonging to the class of antigen-presenting cells. In some embodiments, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are first transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample their environment for pathogens, such as viruses and bacteria. Upon contact with a presentable antigen, they are activated into mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. Concurrently, they upregulate cell surface receptors that act as coreceptors for T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that induces migration of dendritic cells to the spleen via the bloodstream or to lymph nodes via the lymphatic system. Here, they act as antigen-presenting cells by presenting antigens, and activate helper T cells, killer T cells, and B cells in conjunction with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T cell- or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically phagocytose and kill foreign pathogens within the macrophage through hydrolytic and obstetric attack, resulting in their degradation. Peptides from degraded proteins are presented on the macrophage cell surface where they can be recognized by T cells and can directly interact with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

The term "allergen" refers to an antigen that originates outside the subject's body (i.e., an allergen can be referred to as a "foreign antigen") and that causes the subject's immune system to produce an abnormally vigorous immune response to fight off a perceived threat that would otherwise be harmful to the subject. "Allergy" is a disease caused by such a vigorous immune response to an allergen. An allergen is an antigen that can stimulate a type I hypersensitivity reaction, usually via an immunoglobulin E (IgE) response, in atopic individuals. Specific examples of allergens include allergens derived from peanut proteins (e.g., Ara h 2.02), ovalbumin, grass pollen proteins (e.g., Phl p 5), and house dust mite proteins (e.g., Der p 2).

The term "growth factor" refers to a molecule that can stimulate cell growth, proliferation, healing, and/or cell differentiation. Typically, growth factors act as signaling molecules between cells. The term "growth factor" includes certain cytokines and hormones that bind to specific receptors on the surface of target cells. Examples of growth factors include bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), e.g., VEGFA, epidermal growth factor (EGF), insulin-like growth factors, ephrins, macrophage colony-stimulating factors, granulocyte colony-stimulating factors, granulocyte-macrophage colony-stimulating factors, neuregulins, neurotrophic factors (e.g., brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF)), placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS) (anti-apoptotic survival factor), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors (transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β)), and tumor necrosis factor-alpha (TNF-α). In some embodiments, the "growth factor" is a peptide or protein growth factor.

The term "protease inhibitor" refers to a molecule, particularly a peptide or protein, that inhibits the function of a protease. Protease inhibitors are classified by the protease they inhibit (e.g., aspartic protease inhibitors) or by their mechanism of action (e.g., suicide inhibitors, e.g., serpins). Specific examples of protease inhibitors include serpins, such as alpha-1-antitrypsin, aprotinin, and bestatin.

The term "enzyme" refers to a macromolecular biological catalyst that accelerates chemical reactions. Like all catalysts, enzymes are not consumed by the reaction they catalyze and do not alter the equilibrium of that reaction. Unlike many other catalysts, enzymes are much more specific. In some embodiments, enzymes are essential for the homeostasis of a subject; for example, any malfunction of an enzyme (particularly reduced activity, which can be caused by either mutation, deletion, or reduced production) can result in disease. Examples of enzymes include herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminidase, phenylalanine hydroxylase, pseudocholinesterase, and lactase.

The term "receptor" refers to a protein molecule that receives signals from outside the cell (e.g., a chemical signal called a ligand). Binding of a signal (e.g., a ligand) to a receptor causes a certain response in the cell, such as the intracellular activation of a kinase. Receptors include transmembrane receptors (e.g., ion channel-linked (ionotropic) receptors, G protein-linked (metabotropic) receptors, and enzyme-linked receptors) and intracellular receptors (e.g., cytoplasmic receptors and nuclear receptors). Specific examples of receptors include steroid hormone receptors, growth factor receptors, and peptide receptors (i.e., receptors whose ligands are peptides), such as P-selectin glycoprotein ligand-1 (PSGL-1). The term "growth factor receptor" refers to a receptor that binds to a growth factor.

The term "apoptosis regulator" refers to a molecule, particularly a peptide or protein, that regulates apoptosis, i.e., activates or inhibits apoptosis. Apoptosis regulators can be divided into two broad classes: those that regulate mitochondrial function and those that regulate caspases. The first class includes proteins (e.g., BCL-2, BCL-xL) that act to maintain mitochondrial integrity by maintaining mitochondrial membrane potential loss and/or the release of proapoptotic proteins such as cytochrome C into the cytosol. Proapoptotic proteins that promote the release of cytochrome C (e.g., BAX, BAK, BIM) also belong to the first class. The second class includes proteins such as inhibitor of apoptosis proteins (e.g., XIAP) or FLIP, which block caspase activation.

The term "transcription factor" particularly refers to proteins that, by binding to specific DNA sequences, control the rate of transcription of genetic information from DNA to messenger RNA. Transcription factors may govern lifelong cell division, cell growth, and cell death; cell migration and organization during embryonic development; and/or responses to signals from outside the cell, such as hormones. Transcription factors usually contain at least one DNA-binding domain that binds to a specific DNA sequence adjacent to the gene controlled by the transcription factor. Specific examples of transcription factors include MECP2, FOXP2, FOXP3, the STAT protein family, and the HOX protein family.

The term "tumor suppressor protein" refers to a molecule, particularly a peptide or protein, that protects cells from certain pathways leading to cancer. Tumor suppressor proteins (usually encoded by corresponding tumor suppressor genes) exert a depressing or oppressive effect on cell cycle control and/or promote apoptosis. Their functions may be one or more of the following: repression of genes essential for cell cycle continuation; coupling of cell cycle and DNA damage (cell division does not occur as long as damaged DNA is present in the cell); initiation of apoptosis if damaged DNA cannot be repaired; inhibition of metastasis (e.g., preventing tumor cell dispersal, blocking loss of contact inhibition and inhibiting metastasis); and DNA repair. Specific examples of tumor suppressor proteins include p53, phosphatase and tensin homolog (PTEN), SWI/SNF (switch/sucrose nonfermentable), von Hippel-Lindau tumor suppressor (pVHL), adenomatous polyposis coli (APC), CD95, suppressor of tumorigenicity 5 (ST5), suppressor of tumorigenicity 5 (ST5), suppressor of tumorigenicity 14 (ST14), and Yippee-like 3 (YPEL3).

The term "structural protein" refers to a protein that imparts rigidity and stiffness to an otherwise fluid biological component. Structural proteins are mostly fibrous (e.g., collagen and elastin), but can also be globular (e.g., actin and tubulin). Globular proteins are usually soluble as monomers but can polymerize to form long fibers, which can constitute, for example, the cytoskeleton. Other structural proteins are motor proteins that generate mechanical force (e.g., myosin, kinesin, and dynein) and surfactant proteins. Specific examples of structural proteins include collagen, surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D, elastin, tubulin, actin, and myosin.

The term "reprogramming factor" or "reprogramming transcription factor" relates to a molecule, particularly a peptide or protein, that, when expressed in a somatic cell, optionally together with further factors such as further reprogramming factors, leads to the reprogramming or dedifferentiation of said somatic cell into a cell with stem cell characteristics, particularly pluripotency. Specific examples of reprogramming factors include OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG.

The term "genome engineering protein" refers to a protein that can insert, delete, or replace DNA in the genome of a subject. Specific examples of genome engineering proteins include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9).

The term "blood protein" refers to peptides or proteins present in a subject's plasma, particularly the plasma of a healthy subject. Blood proteins have diverse functions, such as transport (e.g., albumin, transferrin), enzymatic activity (e.g., thrombin or ceruloplasmin), clotting (e.g., fibrinogen), defense against pathogens (e.g., complement components and immunoglobulins), and protease inhibitors (e.g., alpha-1-antitrypsin). Specific examples of blood proteins include thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte-colony stimulating factor (G-CSF), modified factor VIII, and anticoagulants.

Thus, in certain embodiments, the pharmaceutically active peptide or protein is (i) a cytokine, preferably selected from the group consisting of erythropoietin (EPO), interleukin 4 (IL-2) and interleukin 10 (IL-11), more preferably EPO; (ii) an adhesion molecule, particularly an integrin; (iii) an immunoglobulin, particularly an antibody; (iv) an immunologically active compound, particularly an antigen, such as a viral or bacterial antigen, for example, an antigen of SARS-CoV-2; (v) a hormone, particularly vasopressin, insulin or growth hormone; (vi) a growth factor, particularly VEGFA; (vii) a protease inhibitor, particularly alpha 1-antitrypsin; (viii) a protease inhibitor, preferably herpes simplex virus type 1 thymidine kinase (HSV1-TK), hexosaminida (ix) enzymes selected from the group consisting of phenylalanine hydroxylase, pseudocholinesterase, pancreatic enzymes, and lactase; (ix) receptors, particularly growth factor receptors; (x) apoptosis regulators, particularly BAX; (xi) transcription factors, particularly FOXP3; (xii) tumor suppressor proteins, particularly p53; (xiii) structural proteins, particularly surfactant protein B; (xiv) reprogramming factors, for example, selected from the group consisting of OCT4, SOX2, c-MYC, KLF4, LIN28, and NANOG; (xv) genome engineering proteins, particularly clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9); and (xvi) blood proteins, particularly fibrinogen.

In some embodiments, the pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or protein to a subject elicits an immune response in the subject against the one or more antigens or one or more epitopes that may be therapeutic or partially or completely protective.

In some embodiments, the nucleic acid (e.g., DNA or RNA, preferably mRNA) encodes at least one epitope, e.g., at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes.

In some embodiments, the target antigen is a tumor antigen and the antigenic sequence (e.g., epitope) is derived from the tumor antigen. Tumor antigens can be "standard" antigens that are generally known to be expressed in a variety of cancers. Tumor antigens can also be "neoantigens" that are specific to an individual's tumor and have not previously been recognized by the immune system. Neoantigens or neo-epitopes can result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. If the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen containing one or more amino acid changes.

Examples of tumor antigens include p53, ART-4, BAGE, beta-catenin/m, Bcr-abl CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, cell surface proteins of the claudin family, such as claudin-6, claudin-18.2 and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, myosin/m, MUC1, M These include, but are not limited to, UM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pml/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, and WT-1.

Cancer mutations are unique to each individual. Therefore, cancer mutations encoding novel epitopes (neo-epitopes) represent attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. Nucleic acids (e.g., DNA or RNA) can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs), resident in the spleen, represent antigen-presenting cells of particular interest for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance the therapeutic efficacy of tumor vaccine compositions. Rapid sequencing of the tumor mutagenesis can provide multiple epitopes for personalized vaccines, which can be encoded, for example, by the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein as a single polypeptide, where the epitopes are optionally separated by linkers. In some embodiments of the invention, a nucleic acid (e.g., DNA or RNA, particularly mRNA) encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include nucleic acids (e.g., DNA or RNA, particularly mRNA) encoding at least five epitopes (designated "pentatopes"), nucleic acids (e.g., DNA or RNA, particularly mRNA) encoding at least 10 epitopes (designated "decatopes"), and nucleic acids (e.g., DNA or RNA, particularly mRNA) encoding at least 20 epitopes (designated "eicosatopes").

In some embodiments, the epitope is derived from a pathogen-associated antigen. In some embodiments, the pharmaceutically active polypeptide and/or antigen or epitope is derived from or is a pathogen protein, an immunogenic variant of the protein, or an immunogenic fragment of the protein or an immunogenic variant thereof.

In some embodiments, the pathogen is selected from viruses, bacteria, fungi, parasites, and other microorganisms.

Exemplary viruses include severe acute respiratory syndrome coronavirus (SARS-CoV), e.g., SARS-CoV2, human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus (CMV) (e.g., CMV5), human herpesvirus (HHV) (e.g., HHV6, 7, or 8), herpes simplex virus (HSV), bovine herpesvirus (BHV) (e.g., BHV4), equine herpesvirus (EHV) (e.g., EHV2), human T-cell leukemia virus (HTLV) 5, and varicella-zoster virus. These include, but are not limited to, varicella zoster (VZV), measles virus, papovavirus (JC and BK), hepatitis virus (e.g., HBV or HCV), myxoma virus, adenovirus, rhinovirus, enterovirus, parvovirus, polyomavirus, influenza virus, papillomavirus (e.g., human papillomavirus (HPV)), poxviruses such as vaccinia virus and molluscum contagiosum virus (MCV), lyssavirus, rotavirus, norovirus, rubella virus, and mumps virus. Exemplary diseases caused by viral infections include, but are not limited to, SARS, acquired immune deficiency syndrome (AIDS), measles, chickenpox, cytomegalovirus infection, genital herpes, hepatitis (e.g., hepatitis B or C), influenza (influenza, e.g., human influenza, swine influenza, canine influenza, equine influenza, and avian influenza), HPV infection, shingles, rabies, the common cold, gastroenteritis, rubella, and mumps.

Exemplary bacteria include, but are not limited to, Campylobacter (e.g., Campylobacter jejuni), Enterobacter species, Enterococcus faecium, Enterococcus faecalis, Escherichia coli (e.g., E. coli O157:H7), Group A Streptococcus, Haemophilus influenzae, Helicobacter pylori, Listeria, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Streptococcus pneumoniae, Salmonella, Shigella, Staphylococcus aureus, Staphylococcus epidermidis, Borrelia and Rickettsia species, Chlamydiaceae, Neisseria gonorrhoeae, Bordetella pertussis, Clostridium tetani, Neisseria meningitidis, Streptococcus (e.g., Streptococcus pneumoniae or Streptococcus pyogenes), and Treponema pallidum. Exemplary diseases caused by bacterial infection include, but are not limited to, anthrax, cholera, diphtheria, food poisoning, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infections, Lyme disease, Rocky Mountain spotted fever, chlamydia, gonorrhea, whooping cough, tetanus, meningitis, scarlet fever, and syphilis.

Exemplary parasites include, but are not limited to, Plasmodium, Trypanosomiasis, Leishmania, Trichomonas, Dientamoeba, Giardia, Entamoeba histolytica, Naegleria, Isospora, Toxoplasma, Sarcocystis, Rhinosporidium sayberi, and Balantidium. Exemplary diseases caused by parasitic infections include, but are not limited to, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, Dientamoebiasis, giardiasis, amebic dysentery, coccidiosis, toxoplasmosis, sarcocystis, rhinosporidiosis, and balantidiosis.

In some embodiments, the pathogen is an infectious pathogen, particularly a pathogen that causes an infectious disease, such as a viral disease, a bacterial disease, or a parasitic disease. In some embodiments, the pathogen is a virus, a bacterium, or a parasite. Thus, in these embodiments, nucleic acids (e.g., DNA or RNA, particularly mRNA) and/or compositions described herein can be used to prevent and/or treat infectious diseases caused by the pathogen.

In one embodiment, the epitope is derived from a viral antigen.

In some embodiments, the antigen or epitope is derived from a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or an immunogenic variant thereof. Thus, in some embodiments, a nucleic acid (e.g., DNA or RNA, particularly mRNA) used in the invention encodes an amino acid sequence comprising a coronavirus protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus protein or an immunogenic variant thereof.

In some embodiments, the antigen or epitope is derived from a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. Thus, in some embodiments, a nucleic acid (e.g., DNA or RNA, particularly mRNA) described herein encodes an amino acid sequence comprising a coronavirus S protein, an immunogenic variant thereof, or an immunogenic fragment of a coronavirus S protein or an immunogenic variant thereof. In some embodiments, the coronavirus is MERS-CoV. In some embodiments, the coronavirus is SARS-CoV. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein are modified RNA, particularly stabilized mRNA. In some embodiments, the RNA contains a modified nucleoside in place of at least one uridine. In some embodiments, the RNA contains a modified nucleoside in place of each uridine. In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In some embodiments, the RNA (particularly mRNA) described herein contains modified nucleosides in place of uridine.

In some embodiments, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

In some embodiments, the RNAs (particularly mRNAs) described herein comprise a 5' cap. In some embodiments, m ₂ ^7,3'-O Gppp(m ₁ ^2'-O ) ApG is utilized as the specific capping structure at the 5' end of the mRNA.

In one embodiment, the RNA (particularly, mRNA) (particularly, vaccine RNA) encoding the antigen comprises a 5' UTR comprising the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:1.

In one embodiment, the RNA (particularly, mRNA) (particularly, vaccine RNA) encoding the antigen comprises a 3' UTR comprising the nucleotide sequence of SEQ ID NO:2 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:2.

In one embodiment, the RNA (particularly, mRNA) (particularly, vaccine RNA) encoding the antigen comprises a polyA sequence. In one embodiment, the polyA sequence comprises at least 100 nucleotides. In one embodiment, the polyA sequence comprises or consists of the nucleotide sequence of SEQ ID NO:3.

In one embodiment of the invention, the antigen (e.g., a tumor antigen or vaccine antigen) is preferably administered as single-stranded, 5'-capped RNA (preferably mRNA) that is translated into the respective protein once it enters the cells of the subject receiving the RNA. Preferably, the RNA contains structural elements (5' cap, 5' UTR, 3' UTR, poly(A) sequence) optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency.

In one embodiment, beta-S-ARCA (D1) is used as the specific capping structure at the 5' end of the RNA. In one embodiment, m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG is used as the specific capping structure at the 5' end of the RNA. In one embodiment, the 5' UTR sequence is derived from human alpha-globin mRNA, optionally with an optimized "Kozak sequence" to increase translation efficiency. In one embodiment, a combination of an "amino-terminal enhancer of split" (AES) mRNA (designated F) and a sequence element (FI element) from mitochondrial-encoded 12S ribosomal RNA (designated I) is placed between the coding sequence and poly(A) sequence to ensure higher peak protein levels and longer mRNA persistence. In one embodiment, two repeated 3' UTRs from human beta-globin mRNA are placed between the coding sequence and poly(A) sequence to ensure higher peak protein levels and longer mRNA persistence. In one embodiment, a poly(A) sequence up to 110 nucleotides in length is used, consisting of 30 adenosine residues followed by a 10 nucleotide linker sequence and a stretch of another 70 adenosine residues, designed to enhance RNA stability and translation efficiency.

Generally, the vaccine RNAs described herein have, from 5' to 3', the following structure:
cap-5' UTR-vaccine antigen-encoding sequence-3' UTR-poly(A)
or beta-S-ARCA(D1)-hAg-Kozak-vaccine antigen-encoding sequence-FI-A30L70
It may include one of:

The RNA or the RNA encoding the vaccine antigen may be unmodified uridine-containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), or self-amplifying RNA (saRNA). In some embodiments, the RNA or the RNA encoding the vaccine antigen is nucleoside-modified mRNA (modRNA).

Unmodified uridine messenger RNA (uRNA)
The active component of unmodified messenger RNA (uRNA) is a single-stranded mRNA that is translated once inside a cell. In addition to the sequence encoding the vaccine antigen (i.e., open reading frame), each uRNA preferably contains consensus structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum RNA effectiveness with respect to stability and translation efficiency. A preferred 5' cap structure is beta-S-ARCA(D1)(m ₂ ^7,2'-O GppSpG). Preferred 5' UTR and 3' UTR comprise the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively. A preferred poly(A) tail comprises the sequence of SEQ ID NO:3.

In this context, "hAg-Kozak" refers to the 5' UTR sequence of human alpha-globin mRNA with an optimized "Kozak sequence" to increase translation efficiency; "FI element" refers to the 3' UTR being a combination of two sequence elements derived from the "amino-terminal enhancer of split" (AES) mRNA (designated F) and the mitochondrially encoded 12S ribosomal RNA (designated I). These were identified through an ex vivo selection process for sequences that confer enhanced RNA stability and total protein expression; "A30L70" refers to a poly(A) tail up to 110 nucleotides long, consisting of 30 adenosine residues, followed by a 10-nucleotide linker sequence and an additional 70 adenosine residues, designed to enhance RNA stability and translation efficiency in dendritic cells; "GS" refers to a sequence encoding a glycine-serine linker, i.e., a short linker peptide consisting primarily of the amino acids glycine (G) and serine (S), commonly used in fusion proteins.

Nucleoside-modified messenger RNA (modRNA)
The active ingredient of nucleoside-modified messenger RNA (modRNA) drug substances is similarly a single-stranded mRNA that is translated once it enters cells. In addition to the sequence encoding the vaccine antigen (i.e., open reading frame), each modRNA, like uRNA, contains consensus structural elements optimized for maximum RNA efficacy (5' cap, 5' UTR, 3' UTR, poly(A) tail). Compared to uRNA, modRNA contains 1-methyl-pseudouridine instead of uridine. The preferred 5' cap structure is m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG. The preferred 5' UTR and 3' UTR contain the nucleotide sequences of SEQ ID NO:1 and SEQ ID NO:2, respectively. The preferred poly(A) tail contains the sequence of SEQ ID NO:3. Additional purification steps are applied to modRNA to reduce dsRNA impurities produced during the in vitro transcription reaction.

Self-amplifying RNA (saRNA)
The active ingredient of a self-amplifying mRNA (saRNA) drug substance is a single-stranded RNA that self-amplifies upon entry into a cell, and the vaccine antigen is subsequently translated. In contrast to uRNA and modRNA, which preferably encode a single protein, the coding region of saRNA contains two open reading frames (ORFs). The 5'-ORF encodes an RNA-dependent RNA polymerase, such as the Venezuelan equine encephalitis virus (VEEV) RNA-dependent RNA polymerase (replicase). The replicase ORF is followed 3' by a subgenomic promoter and a second ORF encoding the antigen. Furthermore, the saRNA UTR contains 5' and 3' conserved sequence elements (CSEs) necessary for self-amplification. Like uRNA, saRNA contains consensus structural elements (5' cap, 5' UTR, 3' UTR, poly(A) tail) optimized for maximum RNA efficacy. saRNA preferably contains a uridine. A preferred 5' cap structure is beta-S-ARCA (D1) (m ₂ ^7,2'-O GppSpG).

Cytoplasmic delivery of saRNA initiates an alphavirus-like life cycle. However, saRNA does not encode alphavirus structural proteins required for genome packaging or cell entry, and therefore, production of replication-competent viral particles is highly unlikely to be possible. Replication does not involve any intermediate steps that produce DNA. Use/uptake of saRNA therefore does not pose the risk of genome integration or other permanent genetic modifications in target cells. Furthermore, saRNA itself efficiently activates the innate immune response through recognition of dsRNA intermediates, thereby preventing its persistent replication.

In addition, a secretory signal peptide (sec) can be fused to the antigen-encoding region, preferably such that sec is translated as an N-terminal tag. In one embodiment, sec corresponds to the secretory signal peptide of the S protein. A sequence encoding a short peptide linker consisting primarily of the amino acids glycine (G) and serine (S), commonly used in fusion proteins, can also be used as the GS/linker.

In some embodiments, a nucleic acid (e.g., DNA or RNA, preferably mRNA) encoding an antigen (e.g., a tumor antigen or vaccine antigen) is expressed in cells of a subject being treated to provide the antigen. In some embodiments, the nucleic acid (e.g., DNA or RNA) is transiently expressed in the subject's cells. In some embodiments, the RNA is transcribed in vitro. In some embodiments, expression of the antigen is at the cell surface. In some embodiments, the antigen is expressed and presented in the context of MHC. In some embodiments, expression of the antigen is in the extracellular space, i.e., the antigen is secreted.

An antigen molecule or its advanced product, e.g., a fragment thereof, can bind to an antigen receptor, such as a BCR or TCR, or an antibody carried by an immune effector cell.

Peptide and protein antigens (wherein the antigen is a vaccine antigen) provided to a subject according to the present invention by administration of nucleic acid (e.g., DNA or RNA, particularly mRNA) encoding the peptide and protein antigen preferably result in the induction of an immune response, e.g., a humoral and/or cellular immune response, in the subject provided with the peptide or protein antigen. The immune response is preferably directed against the target antigen. Thus, a vaccine antigen may comprise the target antigen, a variant thereof, or a fragment thereof. In certain embodiments, such a fragment or variant is immunologically equivalent to the target antigen. In the present invention, the term "antigen fragment" or "antigen variant" refers to an agent that results in the induction of an immune response targeted by the antigen, i.e., the target antigen. Thus, a vaccine antigen may correspond to or comprise a target antigen, a fragment of the target antigen, or an antigen that is homologous to the target antigen or its fragment. Thus, according to the present invention, a vaccine antigen may comprise an immunogenic fragment of the target antigen or an amino acid sequence that is homologous to an immunogenic fragment of the target antigen. According to the present invention, an "immunogenic antigen fragment" preferably refers to a fragment of an antigen capable of inducing an immune response against the target antigen. The vaccine antigen may be a recombinant antigen.

The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and/or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the present invention, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when the amino acid sequence is exposed to a subject's immune system, it induces an immune response with specificity reactive with the reference amino acid sequence. Thus, in certain embodiments, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties as the antigen targeted by T cells and/or exerts the same or essentially the same effect with respect to stimulating, priming, and/or expanding T cells.

In some embodiments, the RNA (preferably mRNA) used in the present invention is non-immunogenic. RNA encoding an immunostimulatory factor can be administered in accordance with the present invention to provide an adjuvant effect. The RNA encoding the immunostimulatory factor can be standard RNA or non-immunogenic RNA.

As used herein, the term "non-immunogenic RNA" (e.g., "non-immunogenic mRNA") refers to RNA that, upon administration to a mammal, does not induce a response by the immune system or that differs only in that it has not been modified to cause the non-immunogenic RNA to be non-immunogenic, e.g., induces a weaker response than that induced by the same RNA, i.e., standard RNA (stdRNA). In certain embodiments, non-immunogenic RNA, which may also be referred to herein as modified RNA (modRNA), is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and/or by limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA) during in vitro transcription, and/or by removing double-stranded RNA (dsRNA) after in vitro transcription, e.g., by the incorporation of modified nucleosides that inhibit RNA-mediated activation of innate immune receptors. In some embodiments, the non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides into the RNA that inhibit RNA-mediated activation of innate immune receptors and/or by removing double-stranded RNA (dsRNA), e.g., after in vitro transcription.

To make non-immunogenic RNA (especially mRNA) non-immunogenic by incorporating modified nucleosides, any modified nucleoside can be used as long as it reduces or suppresses the immunogenicity of RNA. Particularly preferred are modified nucleosides that suppress RNA-mediated activation of innate immune receptors. In some embodiments, the modified nucleoside comprises one or more uridines substituted with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In certain embodiments, nucleosides comprising modified nucleobases are 3-methyl-uridine (m ³ U), 5-methoxy-uridine (mo ⁵ U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s ² U), 4-thio-uridine (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho ⁵ U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm ^{5 U)} , U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s ² U), 5-aminomethyl-2-thio-uridine (nm5s ² U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm5se ² U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s ^{2 U} ), U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm ⁵ U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s ² U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m ⁵ Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m ³ In certain particularly preferred embodiments, the nucleoside comprising a modified nucleobase is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (mψ), or 5-methyl-uridine (m5U), particularly N1-methyl-pseudouridine.

In some embodiments, substitution of one or more uridines with nucleosides comprising modified nucleobases includes substitution of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of uridines.

During the synthesis of RNA (preferably mRNA) by in vitro transcription (IVT) using T7 RNA polymerase, aberrant products containing significant amounts of double-stranded RNA (dsRNA) are produced by the specific activity of the enzyme. dsRNA induces inflammatory cytokines, activates effector enzymes, and leads to the inhibition of protein synthesis. The formation of dsRNA can be limited during the synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) used during synthesis. If desired, UTP can be added once or several times during mRNA synthesis. Alternatively, dsRNA can be removed from RNA, such as IVT RNA, by ion-pair reversed-phase HPLC using, for example, a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, an enzyme-based method using E. coli RNase III can be used to specifically hydrolyze dsRNA but not ssRNA, thereby eliminating dsRNA contamination from the IVT RNA preparation. Additionally, dsRNA can be separated from ssRNA using a cellulosic material. In one embodiment, an RNA preparation is contacted with the cellulosic material, and the ssRNA is separated from the cellulosic material under conditions that allow binding of the dsRNA to the cellulosic material but not ssRNA to the cellulosic material. Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.

"Removing" or "removal," as the term is used herein, refers to the characterization of a population of a first substance, such as non-immunogenic RNA, that is separated from a nearby population of a second substance, such as dsRNA, where the population of the first substance is not necessarily devoid of the second substance and the population of the second substance is not necessarily devoid of the first substance. However, a population of a first substance characterized by the removal of a population of a second substance will have a measurably lower content of the second substance compared to a mixture in which the first and second substances have not been separated.

In certain embodiments, removal of dsRNA (particularly dsmRNA) from non-immunogenic RNA includes removal of dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA. In certain embodiments, the non-immunogenic RNA (particularly mRNA) is free of or essentially free of dsRNA. In certain embodiments, the non-immunogenic RNA (particularly mRNA) composition comprises a purified preparation of single-stranded nucleoside-modified RNA. For example, in certain embodiments, a purified preparation of single-stranded nucleoside-modified RNA (particularly mRNA) is substantially free of double-stranded RNA (dsRNA). In certain embodiments, the purified preparation is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, at least 99.993%, at least 99.994%, at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single-stranded nucleoside-modified RNA relative to all other nucleic acid molecules (e.g., DNA, dsRNA).

A variety of methods can be used to determine the amount of dsRNA. For example, the sample can be contacted with a dsRNA-specific antibody, and the amount of antibody that binds to the RNA can be taken as a measure of the amount of dsRNA in the sample. A sample containing a known amount of dsRNA can be used as a reference.

For example, RNA can be blotted onto a membrane, such as a nylon blotting membrane. The membrane can be blocked, for example, with TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCl, 0.1% (v/v) TWEEN-20) containing 5% (w/v) nonfat dry milk. To detect dsRNA, the membrane can be incubated with a dsRNA-specific antibody, for example, a dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). For example, after washing with TBS-T, the membrane can be incubated with a secondary antibody, for example, HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody can be detected.

In some embodiments, non-immunogenic RNA (particularly mRNA) is translated more efficiently in cells than standard RNA of the same sequence. In some embodiments, translation is enhanced 2-fold relative to its unmodified counterpart. In some embodiments, translation is enhanced 3-fold. In some embodiments, translation is enhanced 4-fold. In some embodiments, translation is enhanced 5-fold. In some embodiments, translation is enhanced 6-fold. In some embodiments, translation is enhanced 7-fold. In some embodiments, translation is enhanced 8-fold. In some embodiments, translation is enhanced 9-fold. In some embodiments, translation is enhanced 10-fold. In some embodiments, translation is enhanced 15-fold. In some embodiments, translation is enhanced 20-fold. In some embodiments, translation is enhanced 50-fold. In some embodiments, translation is enhanced 100-fold. In some embodiments, translation is enhanced 200-fold. In some embodiments, translation is enhanced 500-fold. In some embodiments, translation is enhanced 1000-fold. In some embodiments, translation is enhanced 2000-fold. In some embodiments, the fold increase is between 10- and 1000-fold. In some embodiments, the fold increase is 10-100 fold. In some embodiments, the fold increase is 10-200 fold. In some embodiments, the fold increase is 10-300 fold. In some embodiments, the fold increase is 10-500 fold. In some embodiments, the fold increase is 20-1000 fold. In some embodiments, the fold increase is 30-1000 fold. In some embodiments, the fold increase is 50-1000 fold. In some embodiments, the fold increase is 100-1000 fold. In some embodiments, the fold increase is 200-1000 fold. In some embodiments, translation is enhanced by any other equivalent amount or range of amounts.

In some embodiments, non-immunogenic RNA (particularly mRNA) exhibits significantly less natural immunogenicity than standard RNA having the same sequence. In some embodiments, non-immunogenic RNA (particularly mRNA) exhibits 2-fold less natural immune response than its unmodified counterpart. In some embodiments, natural immunogenicity is reduced 3-fold. In some embodiments, natural immunogenicity is reduced 4-fold. In some embodiments, natural immunogenicity is reduced 5-fold. In some embodiments, natural immunogenicity is reduced 6-fold. In some embodiments, natural immunogenicity is reduced 7-fold. In some embodiments, natural immunogenicity is reduced 8-fold. In some embodiments, natural immunogenicity is reduced 9-fold. In some embodiments, natural immunogenicity is reduced 10-fold. In some embodiments, natural immunogenicity is reduced 15-fold. In some embodiments, natural immunogenicity is reduced 20-fold. In some embodiments, natural immunogenicity is reduced 50-fold. In some embodiments, natural immunogenicity is reduced 100-fold. In some embodiments, natural immunogenicity is reduced 200-fold. In one embodiment, the natural immunogenicity is reduced 500-fold. In one embodiment, the natural immunogenicity is reduced 1000-fold. In one embodiment, the natural immunogenicity is reduced 2000-fold.

The term "exhibiting significantly reduced natural immunogenicity" refers to a detectable reduction in natural immunogenicity. In one embodiment, the term refers to a reduction such that an effective amount of non-immunogenic RNA (particularly mRNA) can be administered without eliciting a detectable natural immune response. In one embodiment, the term refers to a reduction such that the non-immunogenic RNA (particularly mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA. In one embodiment, the reduction is such that the non-immunogenic RNA (particularly mRNA) can be repeatedly administered without eliciting a natural immune response sufficient to eliminate production of the protein encoded by the non-immunogenic RNA.

In some embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein are formulated or are formulated as a liquid, a solid, or a combination thereof.

In some embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein are formulated or will be formulated for injection.

In some embodiments, the nucleic acids (e.g., DNA or RNA, particularly mRNA) described herein are formulated or will be formulated for intramuscular administration.

In certain embodiments, the nucleic acids described herein (e.g., DNA or RNA, particularly mRNA) are formulated or are formulated as compositions, e.g., nucleic acid compositions, particularly pharmaceutical compositions.

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a polymer conjugate compound as defined herein (i.e., an amphiphilic OEG conjugate compound as defined herein, e.g., a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'). ), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (I Xf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), ( V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-2) 3), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39) ), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25).

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid selected from the group consisting of DODMA, DOTMA, DPL14, 3D-P-DMA, Formula (X), and Formula (XI); (iii) a polymer conjugate compound as defined herein (i.e., an amphiphilic OEG conjugate compound as defined herein, e.g., Formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb ), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4) , (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), ( V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22) , (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V- 31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), ( (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably a compound of any of formulas (V-1), (V-5), (V-17), and (V-25); and (iv) one or more additional lipids. In some embodiments, the one or more additional lipids are selected from neutral lipids and combinations thereof. In some embodiments, the neutral lipids include phospholipids, steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination of phospholipids and steroid lipids.

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any of Formulas A-G; or (iii) a polymer conjugate compound as specified herein (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., a compound of Formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), or (Vh'). , (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), ( IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8) ), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V- 25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33 ), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), Any of the compounds of formulae (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25); (iv) phospholipids; and (v) cholesterol.

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (X); (iii) a polymer conjugate compound as specified herein (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., a polymer conjugate compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (V i), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg ), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-2 5), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33) , (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), ( (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25); (iv) phospholipids; and (v) cholesterol.

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (XI) (e.g., any of formulas (XIIa), (XIIb), (XIIIa), (XIIIb), (XIV-1), (XIV-2), and (XIV-3)); (iii) a polymer conjugate compound as specified herein (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (V d'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa' ), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V -3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), ( V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30) , (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39 ), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25); (iv) phospholipids; and (v) cholesterol.

In some embodiments, the composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any of formulas (XV-1) to (XV-6); (iii) a polymer conjugate compound as specified herein (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh) ), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (VIIIa), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), ( IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7) , (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V- 16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24) , (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V -33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41 ), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25); (iv) phospholipids; and (v) cholesterol.

In one embodiment, the nucleic acid is DNA.

In one embodiment, the nucleic acid is RNA, particularly mRNA.

In one embodiment, the composition, particularly the pharmaceutical composition, is a vaccine.

In some embodiments, the composition, particularly the pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

In some embodiments, the nucleic acid (e.g., DNA or RNA) and/or composition, particularly a pharmaceutical composition, is a component of a kit.

In some embodiments, the kit further includes instructions for using the nucleic acid (e.g., DNA or RNA) to induce an immune response in a subject against a pathogen, e.g., a virus such as a coronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments, the kit further includes instructions for use of the nucleic acid (e.g., DNA or RNA) for therapeutic or prophylactic treatment of an infection in a subject, e.g., a viral infection, such as a coronavirus infection. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.

In one embodiment, the subject is a human.

In certain embodiments of the present invention, the nucleic acid (e.g., DNA or RNA) in the nucleic acid (e.g., DNA or RNA) compositions described herein (e.g., in nucleic acid (e.g., DNA or RNA) particles) is at a concentration of about 0.002 mg/mL to about 5 mg/mL, about 0.002 mg/mL to about 2 mg/mL, about 0.005 mg/mL to about 2 mg/mL, about 0.01 mg/mL to about 1 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, or about 0.1 mg/mL to about 0.5 mg/mL. In specific embodiments, the nucleic acid (e.g., DNA or RNA) is at a concentration of about 0.005 mg/mL to about 0.1 mg/mL, about 0.005 mg/mL to about 0.09 mg/mL, about 0.005 mg/mL to about 0.08 mg/mL, about 0.005 mg/mL to about 0.07 mg/mL, about 0.005 mg/mL to about 0.06 mg/mL, or about 0.005 mg/mL to about 0.05 mg/mL.

The particles described herein comprise nucleic acids such as DNA or RNA, particularly mRNA, and can be prepared by subjecting the nucleic acids to a process comprising: (i) nucleic acid, (ii) at least one cationic or cationically ionizable compound; and (iii) (a) a polymer comprising a structure of formula (I) and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., formulas (V), (V′), (Va), (Va′), (Vb), (Vb′), (Vc), (Vc′), (Vd), (Vd′), (Ve), (Ve′), (Vf), (Vf′), (Vg), (Vg′), (Ve′), (Ve′), (Ve′), (Vf ... Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), ( IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), ( V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16) , (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V- 25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33) , (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V- (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably a compound of any of formulas (V-1), (V-5), (V-17) and (V-25). In some embodiments, the particles comprise: (i) nucleic acids (e.g., RNA, particularly mRNA); (ii) at least one cationic or cationically ionizable compound disclosed herein; (iii) (a) a polymer comprising a structure of Formula (I); and (b) a polymer conjugate compound disclosed herein comprising one or more hydrophobic chains (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., Formulas (V), (V′), (Va), (Va′), (Vb), (Vb′), (Vc), (Vc′), (Vd), (Vd′), (Ve), (Ve′), (Vf) , (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc ), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), ( V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V- 15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V -24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41) (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formula (V-1), (V-5), (V-17) and (V-25); (iv) a steroid disclosed herein; and (v) a neutral lipid disclosed herein.

Various types of nucleic acid (e.g., DNA or RNA)-containing particles have previously been described as suitable for delivery of nucleic acids (e.g., DNA or RNA) in particulate form (see, e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid (e.g., DNA or RNA) delivery vehicles, nanoparticle encapsulation of nucleic acids (e.g., DNA or RNA) physically protects the nucleic acid from degradation and, depending on the specific chemistry, may aid in cellular uptake and endosomal escape.

Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to complex formation and spontaneous formation of nucleic acid particles.

During the manufacturing process, introduction of an aqueous solution of nucleic acid (e.g., RNA) into an organic (e.g., ethanolic) solution containing a lipid mixture containing cationically ionizable lipids, e.g., at pH 5, leads to electrostatic interactions between the negatively charged nucleic acid (e.g., DNA or RNA) drug substance and the positively charged cationically ionizable lipids. This electrostatic interaction results in efficient encapsulation of the nucleic acid (e.g., DNA or RNA) drug substance simultaneously with particle formation. After nucleic acid encapsulation, adjusting the medium surrounding the resulting nucleic acid particles (e.g., RNA-LNPs) to, e.g., pH 7-8, neutralizes the surface charge of the particles (e.g., LNPs). When all other variables are held constant, charge-neutral particles exhibit longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are more rapidly cleared within the retina. Upon endosomal uptake, the low pH of the endosomes renders the particles (e.g., LNPs) fusogenic, allowing the release of the nucleic acid (e.g., RNA) into the cytosol of target cells.

In the present invention, the term "particle" refers to a structure formed by molecules or molecular complexes, particularly particle-forming compounds. In certain embodiments, a particle comprises an envelope (e.g., one or more layers or lamellae) composed of one or more types of amphiphilic substances (e.g., amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides). In this context, the expression "amphiphilic substance" refers to a substance that possesses both. The envelope may also comprise additional substances (e.g., additional lipids and/or additional polymers) that do not necessarily have amphiphilic properties. Thus, particles may be monolamellar or multilamellar structures, in which the substances that make up one or more layers or lamellae comprise one or more types of amphiphilic substances (e.g., selected from the group consisting of amphiphilic lipids, amphiphilic polymers, and/or amphiphilic proteins/polypeptides), optionally in combination with additional substances (e.g., additional lipids and/or additional polymers) that do not necessarily have amphiphilic properties. In certain embodiments, the term "particle" refers to a micro- or nano-sized structure, e.g., a small micro- or nano-sized structure. In this context, the term "microsized" means that all three external dimensions of the particle are on the micron scale, i.e., between 1 and 5 μm.

According to the present invention, the term "particle" includes lipoplex particles (LPX), lipid nanoparticles (LNP), polyplex particles, lipopolyplex particles, virus-like particles (VLP), and mixtures thereof (e.g., mixtures of two or more types of particles, e.g., a mixture of LPX and VLP or a mixture of LNP and VLP).

"Nucleic acid particles" can be used to deliver nucleic acids (e.g., RNA, particularly mRNA) to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid particles comprise at least one cationic or cationically ionizable lipid, (a) a polymer comprising the structure of formula (I), and (b) one or more hydrophobic chains (i.e., amphiphilic OEG conjugate compounds as specified herein, e.g., compounds of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI ), (VI’), (VII), (VII’), (VIII), (VIII’), (VIIIa), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), ( IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11) , (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), ( V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V- (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formula (V-1), (V-5), (V-17) and (V-25); and nucleic acids. Nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, liposome-based, polyplex-based, lipopolyplex-based, virus-like particle (VLP)-based formulations, and mixtures thereof. In some embodiments, the nucleic acid particles include lipid nanoparticle (LNP)-based, lipoplex (LPX)-based, and/or liposome-based formulations.

While not intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid and the polymer conjugate compounds disclosed herein, and, if present, additional lipids, combine together with the nucleic acid to form aggregates, which result in colloidally stable particles.

In some embodiments, the particle comprises an amphipathic lipid described herein, particularly a cationic or cationically ionizable amphipathic lipid, and nucleic acid (e.g., RNA, particularly mRNA). In some embodiments, the particle comprises a cationic/cationically ionizable lipid (particularly a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of structures A-G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein); a helper lipid, such as a neutral lipid (e.g., a phospholipid) and a steroid (e.g., cholesterol), and combinations thereof; and an amphipathic OEG conjugate compound specified herein, e.g., a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj ... Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (V h), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), ( VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V -10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V -19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V -28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V -37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably comprising or consisting of any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25).

In some embodiments, the nucleic acid (e.g., RNA) particles disclosed herein comprise (i) a nucleic acid (e.g., RNA, particularly mRNA) described herein; (ii) an amphipathic lipid, particularly a cationic or cationically ionizable amphipathic lipid (e.g., a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of structures A-G disclosed herein; a cationically ionizable lipid of formula (XI) disclosed herein; or a cationic lipid disclosed herein); (iii) (a) a polymer comprising a structure of formula (I) and (b) one or more hydrophobic chains (i.e., an amphipathic OEG conjugate compound as specified herein). compounds, such as formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve') , (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VI I), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IX q), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), ( (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17), and (V-25); (iv) a steroid (e.g., cholesterol); and (v) a neutral lipid (e.g., a phospholipid). In some embodiments, the steroid is cholesterol; and the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE. In some embodiments, the amphiphilic OEG conjugate compound has the formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VII Ia’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp) , (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14) ), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V -43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25). In some embodiments, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the amphiphilic OEG conjugate compound is represented by the formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI' ... VI’), (VII), (VII’), (VIII), (VIII’), (VIIIa), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IX j), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V- 10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25) ), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), ( Any compound of formula (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any compound of formula (V-1), (V-5), (V-17) and (V-25).

In some embodiments, in the nucleic acid particles (e.g., RNA particles) described herein, the nucleic acid (e.g., RNA, particularly mRNA) is bound by a cationically ionizable lipid (particularly a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of structures A-G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), and in the case of LNPs, occupies the central core of the LNP. In some embodiments, the polymer-conjugated compound, together with a phospholipid, forms the surface of the particle (e.g., LNP). In some embodiments, the particle is substantially free of lipids or lipid-like materials comprising polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeat units. In some embodiments, the particle is substantially free of PEG-lipids having at least 30 consecutive ethylene glycol repeat units and substantially free of polysarcosine-conjugated lipids. In some embodiments, the particles comprise (a) a polymer comprising a structure of Formula (I) and (b) one or more hydrophobic chains (i.e., amphiphilic OEG conjugate compounds as specified herein, e.g., compounds of Formula (V), (V′), (Va), (Va′), (Vb), (Vb′), (Vc), (Vc′), (Vd), (Vd′), (Ve), (Ve′), (Vf), (Vf′), (Vg), (Vg′), (Vh), (Vh′), (Vi), (Vi′), (Vj), (Vj′), (VI), (VI′), (VII ... ’), (VIII), (VIII’), (VIIIa), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm ), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13 ), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-2 9), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-4) (V-1), (V-5), (V-17), and (V-25)). In some embodiments, the surface is substantially free of polymer-conjugated lipids other than polymer-conjugated compounds comprising any of the compounds of formulae (V-1), (V-5), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17), and (V-25). In some embodiments, the surface comprises a bilayer. In some embodiments, cholesterol and cationically ionizable lipids in charged and uncharged forms (particularly cationically ionizable lipids of formula (X) disclosed herein; cationically ionizable lipids having one of structures A-G disclosed herein; or cationically ionizable lipids of formula (XI) disclosed herein) can be distributed throughout a particle such as an LNP.

Generally, lipoplexes (LPX) are obtained by mixing two aqueous phases: one containing nucleic acid (especially RNA) and the other containing a lipid dispersion. In some embodiments, the lipid phase comprises liposomes.

In some embodiments, liposomes are self-closing unilamellar or multilamellar vesicular particles, in which the lamellae comprise a lipid bilayer and the enclosed lumen contains an aqueous phase. A prerequisite for using liposomes in nanoparticle formation is that the lipids in the required mixture are capable of forming a lamellar (bilayer) phase in the aqueous environment in which they are applied.

In some embodiments, liposomes comprise a unilamellar or multilamellar phospholipid bilayer enclosing an aqueous core (also referred to herein as an aqueous lumen). They can be made from materials with polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups. In some embodiments, cationic lipids used in the formulation of liposomes designed for nucleic acid delivery are amphiphilic in nature, consisting of a positively charged (cationic) amine head group attached via glycerol to a hydrocarbon chain or cholesterol derivative.

In some embodiments, lipoplexes are multilamellar liposome-based formulations formed by electrostatic interactions between cationic liposomes and nucleic acids (e.g., DNA or RNA). In some embodiments, the formed lipoplexes have different internal arrangements of molecules that contribute to the conversion of the liposomal structure into small nucleic acid-lipoplexes. In some embodiments, these formulations encapsulate little or no nucleic acid (e.g., DNA or RNA) and are characterized by incomplete uptake of nucleic acids.

In some embodiments, LPX particles comprise amphipathic lipids, particularly cationic or cationically ionizable amphipathic lipids, and a nucleic acid (e.g., RNA, particularly mRNA) described herein. In some embodiments, electrostatic interactions between positively charged liposomes (from one or more amphipathic lipids, particularly cationic or cationically ionizable amphipathic lipids) and negatively charged nucleic acid (e.g., RNA, particularly mRNA) result in complex formation and spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes can generally be synthesized using cationic or cationically ionizable amphipathic lipids, such as the cationically ionizable lipid of formula (I), additional lipids such as DOTMA and/or DODMA, and DSPC. In some embodiments, nucleic acid (e.g., RNA, particularly mRNA) lipoplex particles are nanoparticles.

Generally, lipid nanoparticles (LNPs) are obtained by directly mixing nucleic acid (e.g., RNA) in an aqueous phase with lipids in a phase containing an organic solvent, such as ethanol. In this case, the lipid or lipid mixture can be used to form particles that do not form a lamellar (bilayer) phase with water. In some embodiments, the lipid is a cationically ionizable lipid (particularly a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of structures A-G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid disclosed herein (e.g., cholesterol), a neutral lipid disclosed herein (e.g., phospholipid), and an amphiphilic OEG conjugate compound specified herein (e.g., a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj ... Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), ( Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), ( VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr) , (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10) , (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-1 9), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V -28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), Any compound of formula (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any compound of formula (V-1), (V-5), (V-17) and (V-25).

In some embodiments, the particles described herein comprise a cationically ionizable lipid disclosed herein (particularly a cationically ionizable lipid of formula (X) disclosed herein; a cationically ionizable lipid having one of structures A-G disclosed herein; or a cationically ionizable lipid of formula (XI) disclosed herein), a steroid disclosed herein (e.g., cholesterol), a neutral lipid disclosed herein (e.g., phospholipid), and a polymer conjugate compound disclosed herein (i.e., an amphiphilic OEG conjugate compound specified herein, e.g., formula (V), (V ’), (Va), (Va’), (Vb), (Vb’), (Vc), (Vc’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VI I), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (I Xo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7) , (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V -17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V- 26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-3 5), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25).

In some embodiments, a nucleic acid particle (particularly an RNA particle such as an RNA LNP (e.g., an mRNA particle such as an mRNA LNP)) comprises more than one type of nucleic acid molecule, where the molecular parameters of the nucleic acid molecules may be similar to or different from one another, such as with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions, or other characteristics.

In some embodiments, a nucleic acid (e.g., RNA, e.g., mRNA) described herein can be non-covalently associated with a particle described herein. In some embodiments, the nucleic acid (e.g., RNA, particularly mRNA) can be attached to the outer surface of the particle (surface nucleic acid (e.g., surface RNA, particularly surface mRNA)) and/or can be contained within the particle (encapsulated nucleic acid (e.g., surface RNA, particularly encapsulated mRNA)).

As used herein, "nanoparticle" refers to a particle comprising a nucleic acid (particularly RNA such as mRNA) as described herein and at least one cationic lipid, wherein all three external dimensions of the particle are nanoscale, i.e., at least about 1 nm and less than about 1000 nm (preferably 10-990 nm, e.g., 15-900 nm, 20-800 nm, 30-700 nm, 40-600 nm, or 50-500 nm). Preferably, the longest and shortest axes do not differ significantly. Preferably, the size of a particle is its diameter. Preferably, the particle has an average diameter suitable for intravenous administration.

The nucleic acid particles described herein (particularly RNA particles such as mRNA particles) may exhibit a polydispersity index (PDI) of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the nucleic acid particles may exhibit a polydispersity index ranging from about 0.01 to about 0.4 or from about 0.1 to about 0.3.

In the present invention, the term "lipoplex particle" refers to a particle comprising an amphipathic lipid, particularly a cationic amphipathic lipid, and a nucleic acid (particularly RNA, such as mRNA) as described herein. Electrostatic interactions between positively charged liposomes (from one or more amphipathic lipids, particularly cationic amphipathic lipids) and negatively charged nucleic acid (particularly RNA, such as mRNA) result in complex formation and the spontaneous formation of nucleic acid lipoplex particles. Positively charged liposomes can generally be synthesized using a cationic amphipathic lipid, such as DOTMA, and an additional lipid, such as DOPE or DSPC. In some embodiments, the nucleic acid (particularly RNA, such as mRNA) lipoplex particle is a nanoparticle.

The term "lipid nanoparticles" refers to nanosized lipid-containing particles.

In the present invention, the term "polyplex particle" refers to a particle comprising an amphiphilic polymer, particularly a cationic amphiphilic polymer, and a nucleic acid (particularly RNA such as mRNA) as described herein. Electrostatic interactions between the positively charged cationic amphiphilic polymer and the negatively charged nucleic acid (particularly RNA such as mRNA) result in complex formation and the spontaneous formation of nucleic acid polyplex particles. Positively charged amphiphilic polymers suitable for the formulation of polyplex particles include protamine, polyethyleneimine, poly-L-lysine, poly-L-arginine, and histones. In some embodiments, the nucleic acid (particularly RNA such as mRNA) polyplex particle is a nanoparticle.

The term "lipopolyplex particle" refers to a particle comprising an amphiphilic lipid (particularly a cationic amphiphilic lipid) described herein, an amphiphilic polymer (particularly a cationic amphiphilic polymer) described herein, and a nucleic acid (particularly RNA, such as mRNA) described herein. In some embodiments, the nucleic acid (particularly RNA, such as mRNA) lipopolyplex particle is a nanoparticle.

The term "virus-like particle" (abbreviated herein as VLP) refers to a molecule that closely mimics a virus but does not contain any of the viral genetic material and is therefore non-infectious. Preferably, the VLP comprises a nucleic acid (preferably RNA) as described herein, which is heterologous to the virus from which the VLP is derived. VLPs can be synthesized by expressing individual viral structural proteins, which can then self-assemble into virus-like structures. In some embodiments, a combination of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs can be produced from components of a wide variety of viral families, including hepatitis B virus (HBV) (surface antigen-derived mini-HBV (HBsAg)), parvoviridae (e.g., adeno-associated virus), papillomaviridae (e.g., HPV), retroviridae (e.g., HIV), flaviviridae (e.g., hepatitis C virus), and bacteriophages (e.g., Qβ, AP205).

The term "nucleic acid-containing particle" refers to a particle as described herein to which nucleic acid (particularly RNA such as mRNA) is attached. In this regard, the nucleic acid (particularly RNA such as mRNA) can be attached to the outer surface of the particle (surface nucleic acid (particularly surface RNA such as surface mRNA)) and/or can be contained in the particle (encapsulated nucleic acid (particularly encapsulated RNA such as encapsulated mRNA)).

In some embodiments, the particles described herein have a diameter of about 10 to about 2000 nm, e.g., at least about 15 nm (preferably at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or up to 1900 nm (preferably up to about 1900 nm, up to about 1800 nm, up to about 1700 nm, up to about 1600 nm, up to about 1500 nm, up to about 1400 nm, up to about 1300 nm, up to about 1200 nm, up to about 110 0 nm, up to about 1000 nm, up to about 950 nm, up to about 900 nm, up to about 850 nm, up to about 800 nm, up to about 750 nm, up to about 700 nm, up to about 650 nm, up to about 600 nm, up to about 550 nm or up to about 500 nm), preferably from about 20 to about 1500 nm, for example from about 30 to about 1200 nm, from about 40 to about 1100 nm, from about 50 to about 1000 nm, from about 60 to about 900 nm m, about 70-800 nm, about 80-700 nm, about 90-600 nm or about 50-500 nm or about 100-500 nm, for example 10-1000 nm, 15-500 nm, 20-450 nm, 25-400 nm, 30-350 nm, 40-300 nm, 50-250 nm, 60-200 nm or 70-150 nm, preferably a diameter, i.e., twice the radius, such as twice the cross-sectional radius of gyration (R _g ) value or twice the hydrodynamic radius.

In some embodiments, the particles described herein (e.g., LNP and LPX) have a particle size of about 50 nm to about 1000 nm, about 50 nm to about 800 nm, about 50 nm to about 700 nm, about 50 nm to about 600 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm. m, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 100 nm to about 1000 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm nm, about 100 nm to about 500 nm, about 100 nm to about 450 nm, about 100 nm to about 400 nm, about 100 nm to about 350 nm, about 100 nm to about 300 nm, about 100 nm to about 25 nm 0nm, about 100nm to about 200nm, about 150nm to about 1000nm, about 150nm to about 800nm, about 150nm to about 700nm, about 150nm to about 600nm, about 150nm to about about 500 nm, about 150 nm to about 450 nm, about 150 nm to about 400 nm, about 150 nm to about 350 nm, about 150 nm to about 300 nm, about 150 nm to about 250 nm, about 150 nm It has an average diameter ranging from about 200 nm, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 200 nm to about 700 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, about 200 nm to about 450 nm, about 200 nm to about 400 nm, about 200 nm to about 350 nm, about 200 nm to about 300 nm, or about 200 nm to about 250 nm.

With respect to nucleic acid particles (e.g., RNA lipid particles, particularly RNA LNPs such as mRNA LNPs), the N/P ratio or N/P value refers to the ratio of the number of nitrogen groups (especially positively charged polymeric amine (N = nitrogen) groups) on the lipid to the number of negatively charged phosphate (P) groups on the nucleic acid. This corresponds to the charge ratio, as the nitrogen atom (which is pH dependent) is typically positively charged and the phosphate group is negatively charged. The N/P ratio is pH dependent when charge balance exists. Lipid formulations often produce N/P ratios greater than 4, up to 12, because positively charged nanoparticles are believed to be favorable for transfection. In this case, the nucleic acid (e.g., DNA or RNA) is believed to be fully bound to the nanoparticle.

The nucleic acid particles described herein (particularly RNA particles such as mRNA particles) can be produced using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid and/or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain the nucleic acid particles.

As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles of the mixture are microscopic and have particle sizes between 1 and 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. The term "colloid" may refer only to the particles of the mixture, not the entire suspension.

For the preparation of colloids containing at least one cationic or cationically ionizable lipid, methods commonly used and appropriately adapted for the preparation of liposome vesicles are applicable. The most commonly used methods for the preparation of liposome vesicles share the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to form a thin film on the bottom of a flask. The resulting lipid film is hydrated using a suitable aqueous medium to obtain a liposome dispersion. Further miniaturization steps may be included.

Reverse phase evaporation is another method of film hydration for producing liposomal vesicles and involves the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure yields a milky gel, which then replaces the liposomal suspension.

The term "ethanol injection technique" refers to the process of rapidly injecting an ethanol solution containing lipids into an aqueous solution through a needle. This action disperses the lipids in the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. Generally, the nucleic acid (particularly RNA, such as mRNA) lipoplex particles described herein can be obtained by adding nucleic acid (particularly RNA, such as mRNA) to a colloidal liposome dispersion. Using the ethanol injection technique, such colloidal liposome dispersions are formed, in some embodiments, as follows: lipids, e.g., cationically ionizable lipids (e.g., cationically ionizable lipids of formula (X) disclosed herein; cationically ionizable lipids having one of structures A-G disclosed herein; cationically ionizable lipids of formula (XI) disclosed herein; DOTMA and/or DODMA), and additional lipids (e.g., polymer-conjugated lipids described herein (i.e., amphiphilic OEG-conjugated compounds specified herein, e.g., formulas (V), (V'), (Va), etc.). ), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg’), (Vh), (Vh’), (Vi), (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII) , (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V- 1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V- 12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V -22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41) (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17) and (V-25); neutral lipids (e.g., phospholipids); steroids (e.g., cholesterol); and combinations) are injected into the aqueous solution with stirring. In some embodiments, the nucleic acid (particularly RNA, such as mRNA) lipoplex particles described herein are obtained without using an extrusion process.

The terms "extrusion" or "extrusion molding" refer to the creation of particles with a fixed cross-sectional profile, particularly the reduction of particle size by forcing the particles through a filter with defined pores.

Other organic solvent-free methods may also be used in accordance with the present invention to produce colloids.

Particles (e.g., LNPs) typically consist of four particle-forming components: one or more cationic ionizable or cationic lipids; one or more neutral lipids, such as phospholipids; one or more steroids, such as cholesterol; and a polymer-conjugated lipid, such as a PEG-lipid or a polysarcosine-conjugated lipid (sometimes referred to as a "stealth lipid" or "sterically stabilizing lipid"). Each component is responsible for payload protection and enables effective intracellular delivery. However, due to shortcomings of PEG lipids (which are the most common polymer-conjugated lipids), the particles (e.g., LNPs) of the present invention have a slightly different composition: one or more cationically ionizable or cationic lipids; one or more neutral lipids, such as phospholipids; one or more steroids, such as cholesterol; and (a) a polymer comprising the structure of formula (I) and (b) one or more hydrophobic chains (i.e., amphiphilic OEG-conjugated compounds as specified herein, e.g., those of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'). ’), (Vd), (Vd’), (Ve), (Ve’), (Vf), (Vf’), (Vg), (Vg’), (Vh), (Vh’), (Vi) , (Vi’), (Vj), (Vj’), (VI), (VI’), (VII), (VII’), (VIII), (VIII’), (VIIIa ), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), ( V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), ( V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), ( V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), ( V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), ( and any of the compounds of formula (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably any of the compounds of formula (V-1), (V-5), (V-17), and (V-25). In some embodiments, particles of the present invention (e.g., LNPs) are substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeat units. In some embodiments, particles of the present invention (e.g., LNPs) are substantially free of polysarcosine-conjugated lipids. In some embodiments, particles of the present invention (e.g., LNPs) are substantially free of PEG lipids having at least 30 consecutive ethylene glycol repeat units and substantially free of polysarcosine-conjugated lipids. In some embodiments, particles of the present invention (e.g., LNPs) are substantially free of polymer-conjugated lipids other than the amphiphilic OEG-conjugated compounds disclosed herein. Particles (e.g., LNPs) can be prepared by first rapidly mixing lipids (including amphiphilic OEG-conjugated compounds) dissolved in an organic (e.g., ethanolic) solution with nucleic acid (e.g., RNA or DNA) in an aqueous buffer.

Various types of nucleic acid-containing particles have previously been described as suitable for delivery of nucleic acids in particulate form (see, e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids physically protects the nucleic acid from degradation and, depending on the specific chemistry, may aid in cellular uptake and endosomal escape.

In some embodiments, a nucleic acid (e.g., DNA or RNA), at least one cationic or cationically ionizable lipid described herein, and (a) a polymer comprising a structure of formula (I) and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), ( Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), ( IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), ( V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19 ), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V- 28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45 Compositions (particularly particles such as LNPs) containing the polymer-conjugated compounds disclosed herein, including any of the compounds of formulae (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably any of the compounds of formulae (V-1), (V-5), (V-17), and (V-25), further contain one or more additional lipids.

In some embodiments, compositions (particularly particles such as LNPs) comprising nucleic acid (e.g., RNA), at least one cationic or cationically ionizable lipid described herein, and an amphiphilic OEG conjugate compound are prepared by (a) providing (e.g., manufacturing) a nucleic acid solution comprising water and a first buffer system; (b) providing (e.g., preparing) an organic (e.g., ethanolic) solution comprising at least one cationic or cationically ionizable lipid, an amphiphilic OEG conjugate compound described herein, and, if present, one or more additional lipids (e.g., steroids and/or neutral lipids). (c) mixing the nucleic acid solution prepared (e.g., produced) under (a) with the organic (e.g., ethanolic) solution prepared (e.g., produced) under (b), thereby producing a first intermediate formulation comprising particles (e.g., LNPs) dispersed in a first aqueous phase comprising a first buffer system; and (d) filtering (e.g., dialysis, tangential flow filtration, or diafiltration) and/or diluting the first intermediate formulation prepared under (c) using a final aqueous buffer solution comprising a final buffer system, thereby producing a formulation comprising particles (e.g., LNPs) dispersed in a final aqueous phase comprising a final buffer system. Steps (c) and/or (d) can be followed by one or more steps selected from dilution and filtration, e.g., dialysis, tangential flow filtration, or diafiltration.

In some embodiments, a nucleic acid (e.g., RNA), at least one cationic or cationically ionizable lipid described herein, and (a) a polymer comprising a structure of formula (I) and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) ), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (I Xk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5 ), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16) , (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V- 27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37) , (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V- 48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably compounds of the formulae (V-1), (V-5), (V-17) and (V A composition (particularly a particle such as an LNP) comprising a polymer-conjugated compound disclosed herein, including any of the compounds described in any of the above (a) to (c) is prepared by: (a') providing (e.g., manufacturing) a liposome or colloidal formulation of at least one cationic or cationically ionizable lipid, a polymer-conjugated compound disclosed herein, and, if present, one or more additional lipids in an aqueous phase; (b') providing (e.g., manufacturing) a nucleic acid (e.g., RNA) solution comprising water and a buffer system; and (c') mixing the liposome or colloidal formulation prepared (e.g., manufactured) under (a') with the nucleic acid (e.g., RNA) solution prepared (e.g., manufactured) under (b'). Step (c') may be followed by one or more steps selected from dilution and filtration, such as dialysis, tangential flow filtration, or diafiltration.

The present invention relates to a nucleic acid particle comprising a nucleic acid (e.g., DNA or RNA, particularly an LNP comprising RNA), at least one cationic or cationically ionizable lipid, and (a) a polymer comprising the structure of formula (I) and (b) one or more hydrophobic chains (i.e., an amphiphilic OEG conjugate compound as specified herein, e.g., formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc ), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V -15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32 ), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V- and (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62), and (V-63), preferably any of the compounds of formulas (V-1), (V-5), (V-17), and (V-25). The nucleic acid (e.g., DNA or RNA) particles can contain nucleic acids complexed to the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they are not capable of viral infection of T cells.

Suitable cationic or cationically ionizable lipids form nucleic acid particles and are encompassed by the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that binds with nucleic acid to form a nucleic acid particle. Such components include any component that can be part of a nucleic acid particle.

In some embodiments, a nucleic acid (e.g., RNA) particle (particularly an mRNA particle) comprises more than one type of nucleic acid (e.g., RNA) molecule, where the molecular parameters of the nucleic acid molecules may be similar to one another or may differ, such as with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions, or other characteristics.

In a particle formulation, each nucleic acid (e.g., DNA or RNA) species can be formulated separately as an individual particle formulation. In this case, each individual particle formulation contains one nucleic acid (e.g., DNA or RNA) species. The individual particle formulations can be present in separate entities, e.g., separate containers. Such formulations can be obtained by providing each nucleic acid (e.g., DNA or RNA) species separately (typically each in the form of a nucleic acid-containing solution) with a particle-forming agent, thereby forming particles. Each particle exclusively contains the specific nucleic acid (e.g., DNA or RNA) species provided (individual particle formulation) when the particle is formed. In some embodiments, a composition, such as a pharmaceutical composition, contains more than one individual particle formulation. Each pharmaceutical composition is referred to as a mixed particle formulation. A mixed particle formulation according to the present invention can be obtained by separately forming individual particle formulations and then mixing the individual particle formulations. The mixing step results in a formulation containing a mixed population of nucleic acid-containing particles. The individual particle populations can be combined in a single container containing a mixed population of individual particle formulations. Alternatively, all nucleic acid (e.g., DNA or RNA) species of a pharmaceutical composition can be formulated together as a combined particle formulation. Such formulations are obtained by providing a combined formulation (typically a combined solution) of all nucleic acid (e.g., DNA or RNA) species together with a particle-forming agent, thereby forming particles. In contrast to mixed particle formulations, combined particle formulations typically contain particles containing more than one nucleic acid (e.g., DNA or RNA) species. In combined particle compositions, different nucleic acid (e.g., DNA or RNA) species are typically present together in a single particle.

(a) a polymer comprising the structure of formula (I); and (b) a polymer conjugate compound comprising one or more hydrophobic chains (amphiphilic OEG conjugate compound).
One or more of the particle-forming components described herein, such as polymers, lipids, or lipid-like materials, used in the compositions and particles described herein, include (a) a polymer comprising a structure of formula (I); and (b) a polymer conjugate compound comprising one or more hydrophobic chains:
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester, preferably a substituted amide, an optionally substituted thioamide, or an ester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100.

The polymeric conjugate compounds comprise oligoethylene glycol (OEG) stretches (because in formula (I) z is 2 to 24, e.g., 2 to 10, 2 to 7, or 2 to 5, particularly 2 or 3) which are separated from one another by moieties -X ² -X ¹ -Y-, i.e., optionally substituted amide-C _1-3 alkylene, optionally substituted thioamide-C _1-3 alkylene, or ester-C _1-3 alkylene moieties. Furthermore, in addition to the hydrophilic components of formula (I), the polymeric conjugate compounds comprise one or more hydrophobic chains. Therefore, due to these characteristics, the polymeric conjugate compounds are also referred to herein as "amphiphilic OEG conjugate compounds."

Surprisingly, the inventors have discovered that anti-PEG antibodies do not bind to polymers comprising the structure of formula (I); see, e.g., Example 14. Thus, without wishing to be bound by any theory, it is believed that the specific structure of formula (I) (i.e., OEG stretches separated from each other by the moieties -X ² -X ¹ -Y-) prevents anti-PEG antibodies from binding to this structure and similarly to the amphiphilic OEG conjugate compounds of the present invention.

Furthermore, the inventors have discovered that the amphiphilic OEG conjugate compounds of the present invention can be synthesized in a monodisperse form (e.g., by SPPS), unlike polydisperse PEG. In particular, the structure of formula (I), where ^X1 is -C(O)-, ^X2 is -NH-, z is 2, and n is 14, can be readily synthesized in a monodisperse form using SPPS, having a molecular weight of 2050 Da. In contrast, PEK2k can only be synthesized in a polydisperse form, and therefore has an average molecular weight of approximately 2000 Da.

Furthermore, for example, structures of formula (I) in which ^X1 is -C(O)-, ^X2 is -NH-, z is 2, and n is 14, 15, 16, or 17 have properties (e.g., length and hydrophobicity) similar to PEK2k. Thus, in certain embodiments, the polymer portion of the amphiphilic OEG conjugate compound contributes to imparting stealth properties to particles comprising the amphiphilic OEG conjugate compound (and therefore, such amphiphilic OEG conjugate compounds can replace the widely used PEG lipids as stealth lipids).

In some embodiments of formula (I), ^X2 and ^X1 together are an optionally substituted amide. Thus, in some embodiments, the polymer of the amphiphilic OEG conjugate compound has the structure of the following general formula (Ia) or (Ib):
wherein Y, z, and n are as defined above or below; and each ^R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, _-N3 , _C2-6alkynyl , -COOH, _-NH2 , -NH( _C1-3alkyl ), and -N( _C1-3alkyl ) ₂ .
In certain embodiments, R ¹ is the same in each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In certain embodiments, R ¹ in at least one repeat unit is different from R ¹ in other repeat units (e.g., R 1 is a particular alkyl (e.g., H) for at least one repeat unit and R ¹ is a particular alkyl (e.g., methyl) for at least one different repeat unit). In certain embodiments of Formula (Ia), each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl). In certain embodiments of Formula (Ib), ^each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).

In some embodiments of formula (I), ^X2 and ^X1 together are an optionally substituted thioamide. Thus, in some embodiments, the polymer of the amphiphilic OEG conjugate compound has the following general formula (Ic) or (Id):
wherein Y, z, and n are as defined above or below; and each ^R1 is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, _-N3 , _C2-6alkynyl , -COOH, _-NH2 , -NH( _C1-3alkyl ), and -N( _C1-3alkyl ) ₂ .
In certain embodiments, R ¹ is the same in each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In certain embodiments, R ¹ in at least one repeat unit is different from R ¹ in other repeat units (e.g., R 1 is a particular alkyl (e.g., H) for at least one repeat unit and R ¹ is a particular alkyl (e.g., methyl) for at least one different repeat unit). In certain embodiments of Formula (Ic), each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl). In certain embodiments of Formula (Id), ^each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).

In some embodiments of formula (I), ^X2 and ^X1 together are an ester. Thus, in some embodiments, the polymer of the amphiphilic OEG conjugate compound has the following general formula (Ie) or (If):
wherein Y, z, and n are as defined above or below.
Includes the structure of

In some embodiments of formula (I), ^X2 and ^X1 together are a thioester. Thus, in some embodiments, the polymer of the amphiphilic OEG conjugate compound has the following general formula (Ig), (Ih), (Ii), or (Ij):
wherein Y, z, and n are as defined above or below.
Includes the structure of

In some embodiments, the polymer of the amphiphilic OEG conjugate compound comprises the structure of Formula (Ia), wherein R ¹ at each occurrence (i.e., at each repeat unit) is the same and is hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl). In some embodiments, R ¹ at each occurrence (i.e., at each repeat unit) is the same and is hydrogen or methyl. In some embodiments, R ¹ is hydrogen.

In certain embodiments of any of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), and (Ij), Y is —CH ₂ — or —(CH ₂ ) ₂ —. In certain embodiments, Y is —CH ₂ —.

In certain embodiments of any of Formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), and (Ij), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In certain embodiments, z is 2 to 7. In certain embodiments, z is 2 to 5. In certain embodiments, z is 2 or 3. In certain embodiments, z is 2.

In certain embodiments of any of Formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), and (Ij), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In certain embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In certain embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 14. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, n is 12. In certain embodiments, n is 16.

In certain embodiments of any of Formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), Ig), (Ih), (Ii), and (Ij), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, e.g., 6 to 20 or 6 to 15. In certain embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16). In certain embodiments, z is 2 to 5 (e.g., 2 or 3); and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16).

In certain embodiments of the amphiphilic OEG conjugate compound, the polymer has the following general formula (II):
wherein z and n are as defined above or below; and R ¹ is hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).
Includes the structure of

In some embodiments, ^R1 is the same at each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and ^R1 is a specific alkyl (e.g., methyl) for at least one different repeat unit). In some embodiments of Formula (II), ^R1 is the same at each occurrence (i.e., in each repeat unit) and is hydrogen or methyl. In some embodiments, ^R1 is hydrogen.

In some embodiments of Formula (II), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments of Formula (II), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In some embodiments of Formula (II), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (e.g., 2 or 3); and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16).

In certain embodiments of the amphiphilic OEG conjugate compound, the polymer has the following general formula (III):
wherein n is as defined above; and R ¹ is hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl).
Includes the structure of

In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and ^R1 is a specific alkyl (e.g., methyl) for at least one different repeat unit). In some embodiments of Formula (III), ^R1 is the same in each occurrence (i.e., in each repeat unit) and is hydrogen or methyl. In some embodiments, ^R1 is hydrogen.

In some embodiments of formula (III), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In certain embodiments of the amphiphilic OEG conjugate compound, the polymer has the following general formula (IV):
Includes the structure of

In certain embodiments of the amphiphilic OEG conjugate compound, the polymer has the following general formula (IVa):
Includes the structure of

In certain embodiments of either formula (IV) or (IVa), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In certain embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In certain embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 14. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, n is 12. In certain embodiments, n is 16.

In certain embodiments of the amphiphilic OEG conjugate compounds (particularly with respect to any one of formulae (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (II), (III), (IV) and (IVa)), one or more hydrophobic chains are located at the ^X1 or ^X2 terminus of the polymer.

In some embodiments of the amphiphilic OEG conjugate compound (particularly with respect to any one of Formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (II), (III), (IV), and (IVa)), one or more hydrophobic chains are independently selected from acyclic, preferably linear, hydrocarbyl groups, such as the hydrophobic (e.g., lipophilic) chains of natural lipids. In some embodiments, the hydrocarbyl group has at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl group may be saturated or unsaturated. If the amphiphilic OEG conjugate compound includes two or more hydrophobic chains, these chains may be the same or different. For example, if the amphiphilic OEG conjugate compound includes two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In certain other embodiments, the two hydrophobic chains are different; for example, one may be saturated and the other (mono)unsaturated. Examples of the one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, e.g., hydrocarbyl chains of naturally occurring fatty acids, having at least 8 carbon atoms. In specific examples, the one or more hydrophobic chains include hydrocarbyl chains of caprylic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol, stearic alcohol, arachidyl alcohol, behenic alcohol, lignoceryl alcohol, cerotinoyl alcohol, oleyl alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

In some embodiments of the amphiphilic OEG conjugate compound, a polymer comprising the structure of Formula (I) (e.g., a polymer comprising the structure of any one of Formulae (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (II), (III), (IV), and (IVa)) and one or more hydrophobic chains are linked to each other by a linker comprising at least one functionalized moiety. In some embodiments, the linker comprises a heterocycloalkylene moiety attached to the polymer (directly or through at least one additional bifunctionalized moiety). In some embodiments, the linker comprises an alkylene group and a bifunctionalized moiety, where the bifunctionalized moiety attaches the alkylene group to the one or more hydrophobic chains and the alkylene group is attached to the polymer (directly or through at least one additional bifunctionalized moiety). In some embodiments, the linker comprises an alkylene group and a divalent functionalized moiety, where the divalent functionalized moiety connects the alkylene group to one or more hydrophobic chains, the alkylene group is substituted with at least one monovalent functionalized moiety, and the alkylene group is attached to a polymer (directly or via at least one additional difunctionalized moiety). In some embodiments, the linker comprises a divalent functionalized moiety to which one or more hydrophobic chains are attached and which is attached to a polymer (directly or via at least one additional difunctionalized moiety).

In some embodiments, each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azide, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, or imidate. , imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imide, and amide moieties.

In some embodiments, each divalent functionalized moiety is an ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, or carboxylate. Independently selected from carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

In some embodiments, the amphiphilic OEG conjugate compound comprises one of the following structures (wherein the index "0" means that the respective group is absent, while the index "1" means that the group is present):
(hydrophobic chain) _1-2 -(optionally substituted heterocycloalkylene moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)
[(hydrophobic chain)-(difunctionalized moiety)] _1-2 -(alkylene moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)
(hydrophobic chain) _1-2 -(difunctionalized moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)
[(hydrophobic chain)-(difunctionalized moiety) _{0 or 1} ] _1-2 -(alkylene moiety substituted with at least one monofunctionalized moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)

In some embodiments, the amphiphilic OEG conjugate compound has one of the following formulas:
(hydrophobic chain) _1-2 -(optionally substituted heterocycloalkylene moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)-(end group)
[(hydrophobic chain)-(difunctionalized moiety)] _1-2 -(alkylene moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)-(end group)
(hydrophobic chain) _1-2 -(difunctionalized moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)-(end group)
[(hydrophobic chain)-(difunctionalized moiety) _{0 or 1} ] _1-2 -(alkylene moiety substituted with at least one monofunctionalized moiety)-(at least one further difunctionalized moiety) _{0 or 1} -(polymer)-(end group)

In some embodiments, the amphiphilic OEG conjugate compound comprises a terminal group at the end of the polymer opposite to the end at which one or more hydrophobic chains are located. That is, if one or more hydrophobic chains are located at the ^X1 end of the polymer, the terminal group is located at the ^X2 end of the polymer, while if one or more hydrophobic chains are located at the ^X2 end of the polymer, the terminal group is located at the ^X1 end of the polymer. In some embodiments, the terminal group is a neutral terminal group (e.g., H or unsubstituted alkyl) or a functionalized terminal group (e.g., an alkyl group substituted with one or more of hydroxy, thiol, cyano, azide, and amino). In certain embodiments, the terminal group is ^R3 as defined herein (particularly with respect to one of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), and (IXp)).

In some embodiments, the alkylene moiety substituted with at least one monovalent functionalizing moiety is substituted with one or more (e.g., from 1 up to the maximum number of hydrogen atoms attached to the alkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected monovalent functionalizing moieties.

In some embodiments, the alkylene moiety is C _1-6 -alkylene, such as C _1-3 -alkylene, for example methylene, ethylene or trimethylene.

In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted O-heterocycloalkylene, such as an optionally substituted 4- to 14-membered O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted bicyclic O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted 10-membered O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted chromendiyl moiety or a partially or fully hydrogenated form thereof, such as 3,4-dihydro-2H-chromendiyl, particularly 3,4-dihydro-2H-chromene-2,6-diyl.

In certain embodiments, the heterocycloalkylene moiety is substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) substituents independently selected from the first-level substituents, second-level substituents, and third-level substituents specified herein. In certain embodiments, the heterocycloalkylene moiety is substituted with one _{or more} (e.g., from 1 to the maximum number of hydrogen atoms attached to _the heterocycloalkylene moiety, e.g., ₁ , ₂ , ₃ , ₄ , ₅ , or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) substituents independently selected from C _1-3 alkyl, phenyl, halogen, —CF 3 , —OH, —OCH 3 , —SCH 3 , —NH 2-z (CH ₃ ) z , —C(═O)OH, and —C(═O)OCH 3 (where z is 0, 1, or 2, and C 1-3 alkyl is methyl, ethyl, propyl, or isopropyl). In certain embodiments, such substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF ₃ , e.g., halogen (e.g., F, Cl, or Br) and —CF ₃ .

In certain embodiments, the linker connecting the polymer comprising the structure of Formula (I) (e.g., a polymer comprising the structure of any one of Formulae (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (II), (III), (IV), and (IVa)) and the one or more hydrophobic chains further comprises at least one additional bifunctionalized moiety through which the linker, together with the one or more hydrophobic chains, is attached to the ^X1 terminus or the ^X2 terminus. In certain embodiments, the at least one additional bifunctionalized moiety can further comprise an alkylene moiety (e.g., a _C1-6 alkylene moiety, e.g., a _C1-3 alkylene moiety). In certain embodiments, the at least one further difunctionalized moiety is selected from the group consisting of ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamidate, carbonimidate, carboxylate, carboxymethyl ... The linker is selected from the group consisting of carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties, preferably from the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl and thiocarbonyl, wherein if the linker further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a C _1-6 -alkylene group.

In the case of nucleic acid (e.g., DNA or RNA)-lipid particles, a polymer comprising the structure of formula (I) (e.g., a polymer comprising any one of the structures of formulae (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (II), (III), (IV), and (IVa)) is conjugated, preferably covalently bonded, to one or more hydrophobic chains.

In some embodiments, the end groups of the amphiphilic OEG conjugate compound can be functionalized with one or more molecular moieties that impart certain properties, such as a positive or negative charge or a targeting agent that directs the particle to a specific cell type, collection of cells, or tissue.

A wide variety of suitable targeting agents are known in the art. Non-limiting examples of targeting agents include peptides, proteins, enzymes, nucleic acids, fatty acids, hormones, antibodies, carbohydrates, mono-, oligo- or polysaccharides, peptidoglycans, glycopeptides, and the like. In some embodiments, the targeting agent comprises a member of a targeting pair, such as the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkenes (dienophiles) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific (e.g., pegaptanib-VEGF receptor); arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor. For example, any of several different substances can be used that bind to antibodies on the surface of target cells. Antibodies for targeting cell surface antigens generally exhibit the required specificity for the target. In addition to antibodies, suitable immunoreactive fragments or derivatives, such as Fab, Fab', F(ab')2, or scFv fragments, or single-domain antibodies (e.g., camelid _VHH fragments or nanobodies), can also be used. Many antibody fragments suitable for use in forming targeting mechanisms are already available in the art. Similarly, ligands for any receptor on the surface of target cells can be suitably used as targeting agents. These include any small molecules or biomolecules, natural or synthetic, that specifically bind to cell surface receptors, proteins, or glycoproteins found on the surface of the desired target cells.

In some embodiments, the amphiphilic OEG conjugate compound has the following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ^{21 .} , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and a 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and the 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²¹ is selected from the group consisting of C _1-6 alkyl and a 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and the 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
Includes.

In formula (V), ^R2 is attached to the ^X1 end of the polymer and ^R3 is attached to the ^X2 end of the polymer, and in one mode (V'), ^R2 is attached to the ^X2 end of the polymer and ^R3 is attached to the ^X1 end of the polymer.

In some embodiments of formula (V) or (V'), ^X2 and ^X1 together are an optionally substituted amide. Thus, in some embodiments, the amphiphilic OEG conjugate compounds have the following general formulas (Va), (Va'), (Vb), and (Vb'):
wherein R ² , R ³ , Y, z, and n are as defined above or below; and each R ¹ is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with _{one or more} substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —NH ₂ , —NH(C _1-3 alkyl), and —N(C _1-3 alkyl) 2.
In certain embodiments, R ¹ is the same at each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In certain embodiments of any of Formulas (Va), (Va'), (Vb), and (Vb'), R ¹ in at least one repeat unit is different from R ¹ in the other repeat units (e.g., for at least one repeat unit, R ¹ is a particular alkyl (e.g., H) and for at least one different repeat unit, R ¹ is a particular alkyl (e.g., methyl)). In certain embodiments of any of Formulas (Va), (Va'), (Vb), and (Vb'), each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).

In some embodiments of formula (V) or (V'), ^X2 and ^X1 together are an optionally substituted thioamide. Thus, in some embodiments, the amphiphilic OEG conjugate compounds have the following general formulas (Vc), (Vc'), (Vd), and (Vd'):
wherein R ² , R ³ , Y, z, and n are as defined above or below; and each R ¹ is independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with _{one or more} substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —NH ₂ , —NH(C _1-3 alkyl), and —N(C _1-3 alkyl) 2.
In certain embodiments, R ¹ is the same at each occurrence (i.e., in each repeat unit) (e.g., R ¹ can be H or methyl in each repeat unit). In certain embodiments of any of Formulas (Vc), (Vc'), (Vd), and (Vd'), R ¹ in at least one repeat unit is different from R ¹ in the other repeat units (e.g., for at least one repeat unit, R ¹ is a particular alkyl (e.g., H) and for at least one different repeat unit, R ¹ is a particular alkyl (e.g., methyl)). In certain embodiments of any of Formulas (Vc), (Vc'), (Vd), and (Vd'), each R ¹ is independently hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl, e.g., hydrogen or methyl).

In some embodiments of formula (V) or (V'), ^X2 and ^X1 together are an ester. Thus, in some embodiments, the amphiphilic OEG conjugate compounds have the following general formulas (Ve), (Ve'), (Vf), and (Vf'):
wherein R ² , R ³ , Y, z, and n are as defined above or below.
It has one of the following.

In some embodiments of formula (V) or (V'), ^X2 and ^X1 together are a thioester. Thus, in some embodiments, amphiphilic OEG conjugate compounds have the following general formulas (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), and (Vj'):
wherein R ² , R ³ , Y, z, and n are as defined above or below.
It has one of the following.

In certain preferred embodiments, the amphiphilic OEG conjugate compound has formula (Va) or (Va'), wherein R ¹ at each occurrence (i.e., at each repeat unit) is the same and is hydrogen or C _1-8 alkyl (e.g., hydrogen, methyl, or ethyl). In certain embodiments, R ¹ at each occurrence (i.e., at each repeat unit) is the same and is hydrogen or methyl. In certain embodiments, R ¹ is hydrogen.

In certain embodiments of any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj) and (Vj'), Y is -CH ₂ - or -(CH ₂ ) ₂ -. In certain embodiments, Y is -CH ₂ -.

In some embodiments of any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), and (Vj'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments of any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), and (Vj'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In some embodiments of any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), and (Vj'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (e.g., 2 or 3); and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16).

In certain embodiments of any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj) and (Vj'), Y is -CH ₂ -; z is 2 to 20, for example, 2 to 15, 2 to 10 or 2 to 7; and n is 5 to 25, for example, 6 to 20 or 6 to 15. In certain embodiments, Y is -CH ₂ -; z is 2 to 7; and n is 7 to 16, for example, 7 to 14 (for example, 8, 10, 12, 14 or 16). In some embodiments, Y is —CH ₂ —; z is 2 to 5 (eg, 2 or 3); and n is 7 to 16, for example, 7 to 14 (eg, 8, 10, 12, 14, or 16).

In some embodiments, the amphiphilic OEG conjugate compound has the following general formula (VI) or (VI'):
wherein R ² , R ³ , z, and n are as defined for formulas (V) and (V'); and R ¹ is hydrogen or C _1-8 alkyl.
having

In some embodiments of Formula (VI) or (VI'), ^R1 is hydrogen or methyl. For example, ^R1 can be hydrogen. In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and a specific alkyl (e.g., methyl) for at ^least one different repeat unit).

In some embodiments of formula (VI) or (VI'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments of formula (VI) or (VI'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In some embodiments of formula (VI) or (VI'), z is 2 to 20, e.g., 2 to 15, 2 to 10, or 2 to 7; and n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, z is 2 to 7; and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16). In some embodiments, z is 2 to 5 (e.g., 2 or 3); and n is 7 to 16, e.g., 7 to 14 (e.g., 8, 10, 12, 14, or 16).

In some embodiments, the amphiphilic OEG conjugate compound has the following general formula (VII) or (VII'):
wherein R ² , R ³ and n are as defined for formulas (V) and (V'); and R ¹ is hydrogen or C _1-8 alkyl.
It has.

In some embodiments of Formula (VII) or (VII'), ^R1 is hydrogen or methyl. For example, ^R1 can be hydrogen. In some embodiments, ^R1 is the same in each occurrence (i.e., in each repeat unit) (e.g., ^R1 can be H or methyl in each repeat unit). In some embodiments, ^R1 in at least one repeat unit is different from ^R1 in other repeat units (e.g., ^R1 is a specific alkyl (e.g., H) for at least one repeat unit and a specific alkyl (e.g., methyl) for at ^least one different repeat unit).

In some embodiments of formula (VII) or (VII'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In some embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In some embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 14. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, n is 12. In some embodiments, n is 16.

In some embodiments, the amphiphilic OEG conjugate compounds have the following general formulas (VIII), (VIII'), (VIIIa) and (VIIIa'):
wherein R ² , R ³ and n are as defined for formulas (V) and (V′).
It has one of the following.

In certain embodiments of any of formulas (VIII), (VIII'), (VIIIa), and (VIIIa'), n is 5 to 50, e.g., 5 to 45, 5 to 40, 5 to 35, or 5 to 30. In certain embodiments, n is 5 to 25, e.g., 6 to 20 or 6 to 15. In certain embodiments, n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 14. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, n is 12. In certain embodiments, n is 16.

In some embodiments of the amphiphilic OEG conjugate compound (particularly with respect to any of formulae (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), one or more hydrophobic chains are independently selected from acyclic, preferably linear, hydrocarbyl groups, e.g., hydrophobic (e.g., lipophilic) chains of natural lipids. In certain embodiments, the hydrocarbyl group has at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The hydrocarbyl group may be saturated or unsaturated. If the amphiphilic OEG conjugate compound includes two or more hydrophobic chains, these chains may be the same or different. For example, if the polymer conjugate compound includes two hydrophobic chains, in certain embodiments, the two hydrophobic chains are the same. In certain other embodiments, the two hydrophobic chains are different, e.g., one may be saturated and the other (mono)unsaturated. Examples of the one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, e.g., hydrocarbyl chains of naturally occurring fatty acids, having at least 8 carbon atoms. In specific examples, the one or more hydrophobic chains include the hydrocarbyl chains of caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

In certain embodiments of amphiphilic OEG conjugate compounds having any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa'), R ² is R ⁴ or -L ¹ (R ⁴ ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group (i.e., one of the one or more hydrophobic chains is included in R ² ); L ¹ is a linker; and p is 1 or 2.

In certain embodiments, ^L1 comprises at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or is attached to a divalent functionalized moiety at the terminal where the alkylene group is attached to ^R4 , wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocathion, thiocarbamate ... Carbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemi and/or each divalent functionalized moiety is independently selected from cetal, ketal, hemiketal, imide, and amide moieties; and/or each divalent functionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate , thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), ^L1 comprises an optionally substituted heterocycloalkylene moiety attached to the polymer (directly or through at least one further difunctionalized moiety). In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted O-heterocycloalkylene, such as an optionally substituted 4- to 14-membered O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted bicyclic O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted 10-membered O-heterocycloalkylene. In certain embodiments, the optionally substituted heterocycloalkylene moiety is an optionally substituted partially or fully hydrogenated chromenediyl moiety, such as 3,4-dihydro-2H-chromenediyl, particularly 3,4-dihydro-2H-chromene-2,6-diyl. In certain embodiments, the heterocycloalkylene moiety is substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the heterocycloalkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) substituents independently selected from the first-level substituents, second-level substituents, and third-level substituents specified herein. In certain embodiments, the heterocycloalkylene moiety is substituted with one _{or more} (e.g., from 1 to the maximum number of hydrogen atoms attached to _the heterocycloalkylene moiety, e.g., ₁ , ₂ , ₃ , ₄ , ₅ , or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) substituents independently selected from C _1-3 alkyl, phenyl, halogen, —CF 3 , —OH, —OCH 3 , —SCH 3 , —NH 2-z (CH ₃ ) z , —C(═O)OH, and —C(═O)OCH 3 (where z is 0, 1, or 2, and C 1-3 alkyl is methyl, ethyl, propyl, or isopropyl). In certain embodiments, such substituents are selected from the group consisting of methyl, ethyl, propyl, isopropyl, halogen (e.g., F, Cl, or Br), and —CF ₃ , e.g., halogen (e.g., F, Cl, or Br) and —CF ₃ . In certain embodiments, the optionally substituted heterocycloalkylene moiety is 3,4-dihydro-2H-chromene-2,6-diyl substituted with a methyl group at (i) each of positions 2, 5, 7, and 8 (corresponding to the methyl substitution pattern of α-tocopherol), (ii) each of positions 2, 5, and 8 (corresponding to the methyl substitution pattern of β-tocopherol), (iii) each of positions 2, 7, and 8 (corresponding to the methyl substitution pattern of γ-tocopherol), or (iv) each of positions 2 and 8 (corresponding to the methyl substitution pattern of δ-tocopherol).

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), ^L1 comprises an alkylene group and a difunctionalized moiety, wherein the difunctionalized moiety connects the alkylene group to one or more hydrophobic chains and the alkylene group is connected to the polymer (directly or through at least one additional difunctionalized moiety). In certain embodiments, the alkylene moiety is C _1-6 -alkylene, such as C _1-3 -alkylene, such as methylene, ethylene, or trimethylene. In certain preferred embodiments, the alkylene moiety is trimethylene. In certain embodiments, the divalent functionalized moiety is selected from the group consisting of esters, amides, sulfides, disulfides, sulfones, orthoesters, acylhydrazones, hydrazines, oximes, acetals, and ketals. In certain embodiments, the divalent functionalized moiety is selected from the group consisting of esters and amides.

In certain embodiments (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), L ¹ comprises an alkylene group and a divalent functionalized moiety, where the divalent functionalized moiety connects the alkylene group to one or more hydrophobic chains, the alkylene group is substituted with at least one monovalent functionalized moiety, and the alkylene group is attached to a polymer (directly or via at least one additional difunctionalized moiety). In certain embodiments, the alkylene moiety substituted with at least one monovalent functionalized moiety is substituted with one or more (e.g., from 1 to the maximum number of hydrogen atoms attached to the alkylene moiety, e.g., 1, 2, 3, 4, 5, or 6, e.g., 1-5, 1-4, or 1-3, or 1 or 2) independently selected monovalent functionalized moieties. In certain embodiments, the alkylene moiety is C _1-6 -alkylene, e.g., C _1-3 -alkylene, e.g., methylene, ethylene, or trimethylene. In certain preferred embodiments, the alkylene moiety is trimethylene. In some embodiments, the alkylene moiety is trimethylene substituted with an OH moiety and a hydrophobic chain directly attached to the trimethylene moiety (preferably, the OH moiety and the hydrophobic chain are attached to the same carbon atom of the trimethylene moiety (e.g., one of the terminal carbon atoms)). In some embodiments, the hydrophobic chain directly attached to the trimethylene moiety is unsaturated, preferably monounsaturated. In some embodiments, the hydrophobic chain directly attached to the trimethylene moiety (and preferably monounsaturated) has at least 8 carbon atoms, e.g., at least 10 carbon atoms.

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), ^L1 comprises a divalent functionalized moiety having one or more hydrophobic chains attached thereto and attached to the polymer (directly or via at least one additional difunctionalized moiety). In certain embodiments, the divalent functional group is an amino group. In some embodiments, the divalent functionalized moiety is an unsubstituted amino group (ie, the R ² substituent is —N(R ⁴ ) ₂ ).

In certain embodiments (particularly with respect to any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ](C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(═N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N═)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)-, wherein * is R R _{25 is selected from the group consisting of C 1-6} ^alkyl , aryl, and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl); r is an integer from 1 to 2; ^3,4 -dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN _, and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to the other hydrophobic chain R ⁴ .

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), L ¹ further comprises at least one additional bifunctionalized moiety through which R ² is linked to X ¹ of Formula (V). (or a carbonyl group of any of formulas (Va), (Vb), (Vc), (Vd), (Ve), (Vf), (Vg), (Vh), (Vi), (Vj), (VI), (VII), (VIII) and (VIIIa)) or X ² of formula (V') (or an N atom of any of formulas (Va'), (Vb'), (Vc'), (Vd'), (Ve'), (Vf'), (Vg'), (Vh'), (Vi'), (Vj'), (VI'), (VII'), (VIII') and (VIIIa')). In some embodiments, the at least one additional difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino. (imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably ether, amino, phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl. In certain embodiments, the at least one further difunctionalized moiety is selected from the group consisting of ether and amino. If ^L1 further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a _C1-6 -alkylene group.

In certain embodiments (particularly with respect to any of formulae (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, wherein * is R p is 1 or 2; the C ^1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C _1-6 alkyl, aryl, aryl(C _1-6 alkyl) and a counter cation (e.g., the counter cation can be a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, such as a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _1-6 -alkyltriyl optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ .

In certain embodiments (particularly with respect to any of formulae (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)—, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)—, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-OC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )—O(C _1-6 -alkylene)C(O)—, *-(3,4-dihydro-2H-chromen-6-yl)O—, [*-C(O)O] _p (C _1-6 -alkylene)O—, [*-OC(O)] _p (C _1-6 -alkylene)O—, (*-) ₂ N— and [*-C(O)NH](C _1-6 -alkyltriyl)O—; or L ¹ is (*-)(R ²⁶ )N—, wherein * is R p is 1 or 2; the C ^1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ or an organic cation, for example, a quaternary ammonium or amine cation); 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN and —OC _1-3 alkyl; and C _1-6 -alkyltriyl optionally substituted with one or more —OH substituents and directly bonded to another hydrophobic chain R ⁴ .

In certain embodiments (particularly with respect to any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )—O(C _1-3 -alkylene)NH—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O( ^C _1-3 -alkylene)—, C(O)NH] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O-, (R ⁴ ) ₂ N- and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O- or R ² is (R ⁴ )(R ²⁶ )N—, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation (e.g., the counter cation can be a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, such as a quaternary ammonium or amine cation); 2-R ⁴ -3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl is optionally substituted with one or more —OH substituents, and the other hydrophobic chain R It is directly bonded to ⁴ .

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), ^R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety and a ceramide moiety, or ^R2 is a monoalkylamine moiety.

In certain embodiments, (particularly with respect to any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), each of the one or more hydrophobic chains (i.e., each ^R4 ) is independently an acyclic, preferably linear, hydrocarbyl group, e.g., a hydrophobic (e.g., lipophilic) chain of a naturally occurring lipid. In some embodiments, one or more hydrocarbyl groups independently have at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The one or more hydrocarbyl groups may be saturated or unsaturated. If the amphiphilic OEG conjugate compound contains two or more hydrophobic chains, these chains may be the same or different. For example, if the amphiphilic OEG conjugate compound contains two hydrophobic chains, in some embodiments, the two hydrophobic chains are the same. In some other embodiments, the two hydrophobic chains are different, e.g., one may be saturated and the other (mono)unsaturated, and/or the two hydrophobic chains have different lengths. Examples of the one or more hydrophobic chains include hydrocarbyl chains of fatty acids, particularly hydrocarbyl chains of naturally occurring fatty acids, e.g., hydrocarbyl chains of naturally occurring fatty acids, having at least 8 carbon atoms. In particular embodiments, the one or more hydrophobic chains include hydrocarbyl chains of caprylic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, palmityl alcohol, stearic alcohol, arachidyl alcohol, behenic alcohol, lignoceryl alcohol, cerotinoyl alcohol, oleyl alcohol, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, oleic acid, and tocopherol.

In certain embodiments (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), R ^R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl (e.g., α-tocopheryl, β-tocopheryl, γ-tocopheryl, or δ-tocopheryl), DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or ^R2 is a monomyristylamine moiety.

In certain embodiments (particularly with respect to any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), R ³ is H, C _1-6 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ is selected from the group consisting of a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and a 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair.

In certain embodiments (particularly with respect to any of formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), R ³ is H, C _1-3 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² and a member of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with ^{one or} more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R and R ²² and R ^{23 are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 together with the} nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ and members of a targeting pair.

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), ^R3 is selected from the group consisting of H, -C(O)(Ci _-3 alkyl), -NH(Ci _-3 alkyl), -N(Ci _-3 alkyl) ₂ and a member of a targeting pair, wherein C _{The 1-3} alkyl groups are optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -C(O)NH ₂ , -C(O)NHCH ₃ , -C( _{O)NH(CH 2} ) ₂ NH ₂ and members of a targeting pair.

In certain embodiments, (particularly with respect to any of Formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa')), the targeting pair is selected from the following pairs: maleimide- thiol; thiol-alkyl halide (especially brominated); azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkenes (dienophiles) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor. In certain embodiments, the member of the targeting pair is selected from the group consisting of a maleimide moiety, a thiol moiety, a halogenated (particularly brominated) alkyl moiety, an azide moiety, an alkynyl moiety, an antigen, and an antibody (including a fragment or derivative thereof).

In some embodiments, the amphiphilic OEG conjugate compounds have the following formulae (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr):
wherein n is 5 to 25; R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl), and a member of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH ₃ , —C(O)NH(CH ₂ ) ₂ NH ₂ , and a member of a targeting pair; R ²⁷ is H or a counter cation (e.g., the counter cation is a cation of a pharmaceutically acceptable salt, such as an alkali metal (e.g., sodium or potassium) cation; an alkaline earth metal (e.g., calcium or magnesium) cation; ammonium (NH ₄ ⁺ ); or an organic cation, for example, a quaternary ammonium or amine cation); and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
It has one of the following.

In certain embodiments of any of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), n is 7 to 16, e.g., 7 to 14, preferably 8, 10, 12, 14, or 16.

In certain embodiments of any of formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), the member of the targeting pair is selected from the group consisting of a maleimide moiety, a thiol moiety, a halogenated (particularly brominated) alkyl moiety, an azide moiety, an alkynyl moiety, an antigen, and an antibody (including a fragment or derivative thereof).

In certain embodiments of any of Formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), ^R3 is H or —C(O)(Ci _-3 alkyl), wherein the Ci _-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl(maleimidyl), —SH, —Br, _—N3 , and _C2-6 alkynyl.

In certain embodiments of any of Formulas (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), n is 7 to 16, for example, 7 to 14 (preferably 8, 10, 12, 14, or ₁₆ ); and ^R3 is H or —C(O)(Ci _-3 alkyl), wherein the Ci _-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N3, and _C2-6 alkynyl.

Specific examples of amphiphilic OEG conjugate compounds are the following compounds (V-1) to (V-63):

Particularly preferred examples of amphiphilic OEG conjugate compounds are those having formulae (V-1), (V-5), (V-17), and (V-25) and salts thereof.

An amphiphilic OEG conjugate compound or a polymer conjugate compound comprising (a) a polymer having a structure of formula (I); and (b) one or more hydrophobic chains (particularly, a polymer conjugate compound having a structure of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII ’), (VIIIa), (VIIIa’), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp) , (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16) , (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V -33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49) It is understood that any reference to any compound of formula (V-1), (V-5), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any compound of formula (V-1), (V-5), (V-17) and (V-25), also includes salts (particularly pharmaceutically acceptable salts), tautomers, stereoisomers, solvates (e.g., hydrates) and isotopically labeled forms thereof.

In some cases, amphiphilic OEG conjugate compounds (particularly those of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXh), (IXi), (IXj ... Xf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7) , (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V- 16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24) , (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), ( V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-4 1), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49) , (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V The compound of any of formulas (V-1), (V-5), (V-17) and (V-25)) may comprise from about 0.1 mol% to about 20 mol%, for example from about 0.2 mol% to about 15 mol%, from about 0.5 mol% to about 10 mol%, from about 1 mol% to about 5 mol%, or from about 1.5 mol% to about 2.5 mol%, or from about 2 mol% to about 6 mol%, or from about 2 mol% to about 5 mol%, of the total lipid/particle present in the composition. In some cases, amphiphilic OEG conjugate compounds (particularly those of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI) , (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd) , (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (I Any compound of (Xq) and (IXr) (wherein n is 14), for example, (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-1 The lipids (V-8), (V-19) and (V-20), preferably compounds of any of formulae (V-1), (V-5) and (V-17)) may comprise from about 0.1 mol% to about 2.5 mol%, for example from about 0.2 mol% to about 2.4 mol%, from about 0.5 mol% to about 2.2 mol%, or from about 1 mol% to about 2 mol% of the total lipid/particle present in the composition. In some cases, amphiphilic OEG conjugate compounds (particularly those of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IX f), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq) and (IXr) compounds (wherein n is 8), such as (V-21), (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39) and V-40), preferably a compound of formula (V-25)), may constitute from about 2 mol% to about 5 mol%, for example from about 2.5 mol% to about 5 mol%, in particular from about 3 mol% to about 5 mol%, about 3.5 mol% to about 4.5 mol%, about 3.6 mol% to about 4.4 mol%, about 3.8 mol% to about 4.2 mol% or about 4 mol% of the total lipid/particle present in the composition. In some cases, an amphiphilic OEG conjugate compound (particularly a compound of any of formulae (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa) and (VIIIa') (wherein R The lipids ( ^V-2 containing a ceramide moiety), e.g., (IXo), (IXp), (V-13), (V-14), (V-15), (V-16), (V-33), (V-34), (V-35), (V-36), (V-53), (V-54), (V-55) and (V-56), preferably compounds of formula (V-13)), may constitute from about 2 mol% to about 5 mol%, for example from about 2.5 mol% to about 5 mol%, particularly from about 3 mol% to about 5 mol% of the total lipid/particle present in the composition.

Amphiphilic OEG conjugate compounds can be prepared using conventional synthetic methods known to those skilled in the art. For example, an intermediate compound (e.g., Ac-(AEEA) _n -OH or H-(AEEA) _n -OH; AEEA = (2-(2-(2-aminoethoxy)ethoxy)acetic acid) can be prepared using solid-phase peptide synthesis (SPPS). After isolation and purification of the intermediate compound, it can be conjugated with an organic molecule (e.g., a compound containing a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety) to obtain an amphiphilic OEG conjugate compound. Finally, the amphiphilic OEG conjugate compound can be purified and isolated. The above steps are now described in more detail.

Synthesis of Intermediate Compounds (e.g., Ac-(AEEA) _n -OH or H-(AEEA) _n -OH) The synthesis of intermediate compounds (e.g., Ac-(AEEA) ₁₄ -OH or Ac-(AEEA) ₈ -OH) can be carried out using solid phase peptide synthesis (SPPS). The solid support, also referred to as resin, is chosen to provide a free acid at the C-terminus. The resin is suspended in an organic solvent to ensure proper swelling. The scale of the synthesis depends on the resin substitution and may involve multiple replicates. The synthesis begins with optional deprotection of the resin (e.g., if the amino group on the resin is protected), followed by coupling with the desired amino acid (e.g., Fmoc-AEEA-OH). Coupling is carried out in a suitable solvent mixture containing coupling agents such as, but not limited to, carbodiimide and benzotriazole. After coupling, the solution is drained, and the peptidyl-resin is washed and subjected to Fmoc deprotection/next amino acid coupling until the expected sequence is complete. If applicable (to obtain an N-terminally acetylated intermediate), the final N-terminal Fmoc is first deprotected (to obtain the peptidyl resin H-(AEEA) _n -resin) and then acetylated (to obtain the acetylated peptidyl resin Ac-(AEEA) _n -resin). The intermediate compound is then cleaved from the resin under acidic conditions to obtain crude Ac-(AEEA) _n -OH or H-(AEEA) _n -OH. The solution may then be evaporated, and the crude intermediate may be resolubilized, for example, in an aqueous and organic solvent mixture. The solubilized crude intermediate can be further purified, for example, using a chromatographic system (with or without pre-purification, which may involve solvent exchange and/or salt exchange (e.g., by use of lyophilization)), in particular a liquid chromatographic system (e.g., HPLC), which preferably includes a suitable detector (e.g., a mass detector). Pure relevant fractions can be collected, pooled and lyophilized.

Conjugation of intermediate compounds (especially Ac-(AEEA) _n -OH) with organic molecules to obtain amphiphilic OEG conjugate compounds The lyophilized Ac-(AEEA) _n -OH is solubilized in an organic solvent and reacted with an organic molecule (e.g., a compound containing a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety), preferably in the presence of a coupling agent (e.g., but not limited to, a carbodiimide and/or a benzotriazole), to obtain an amphiphilic OEG conjugate compound (e.g., Ac-(AEEA) ₁₄ -DSPE or Ac-(AEEA) ₁₄ -α-tocopherol). If applicable, the amphiphilic OEG conjugate compounds are purified, for example, using a chromatographic system (with or without pre-purification, where pre-purification may involve solvent exchange and/or salt exchange (e.g., by use of lyophilization)), in particular a liquid chromatographic system (e.g., HPLC), where the chromatographic system preferably includes a suitable detector (e.g., a mass detector). Pure relevant fractions may be collected, pooled and lyophilized.

Conjugation of an intermediate compound (especially H-(AEEA) _n -OH) with an organic molecule to obtain an amphiphilic OEG conjugate compound Preferably, the N-terminus of H-(AEEA) _n -OH is first subjected to a protection reaction (e.g., a BOC protection reaction), and optionally purified (e.g., using a chromatography system) to obtain PG-(AEEA) _n -OH, where PG is an amino protecting group (e.g., PG-(AEEA) _n -OH can be BOC-(AEEA) ₁₄ -OH). PG-(AEEA) _n -OH is then reacted with an organic molecule (e.g., a compound containing a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety), preferably in the presence of a coupling agent (e.g., but not limited to, a carbodiimide and/or a benzotriazole), to yield a PG-(AEEA) _n -organic molecule, i.e., an N-terminal protected version of the amphiphilic OEG conjugate compound (e.g., a Boc-(AEEA) _n -organic molecule, such as Boc-(AEEA) ₁₄ -DSPE or Boc-(AEEA) ₁₄ -α-tocopherol). Preferably, the PG-(AEEA) _n -organic molecule is then subjected to a deprotection reaction to remove the amino protecting group to give the final amphiphilic OEG conjugate compound (e.g., H-(AEEA) ₁₄ -DSPE or H-(AEEA) ₁₄ -α-tocopherol). If applicable, the amphiphilic OEG conjugate compound is purified, for example, using a chromatographic system (with or without pre-purification, where pre-purification may include solvent exchange and/or salt exchange (e.g., by use of lyophilization)), in particular a liquid chromatographic system (e.g., HPLC), where the chromatographic system preferably includes a suitable detector (e.g., a mass detector). Pure relevant fractions may be collected, pooled and lyophilized.

Thus, a method for preparing an amphiphilic OEG conjugate compound may comprise the steps of: (a) providing an intermediate compound having formula (VII) or (VII'), wherein for formula (VII), ^R2 is OH and ^R3 is H, acetyl or Fmoc; and for formula (VII'), ^R2 is H, acetyl or Fmoc and ^R3 is OH; and (b) conjugating the intermediate compound provided under (a) with an organic molecule, particularly a compound comprising a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, thereby obtaining an amphiphilic OEG conjugate compound.

In one embodiment of the method for preparing an amphiphilic OEG conjugate compound, in step (a), an intermediate compound having formula (VII) or (VII') is prepared using solid phase peptide synthesis (SPPS) and the corresponding N-terminally protected AEEA-containing compound (e.g., Fmoc-AEEA-OH). For example, this preparation by SPPS may include the following steps: (a1) suspending a solid phase (e.g., a resin) bearing an amino group in a suitable organic solvent (preferably to ensure adequate swelling of the solid phase); (a2) optionally, if the amino group of the solid phase is protected, performing a deprotection reaction (e.g., PG-solid phase (where PG is an amino-protecting group) with H ₂ N-solid phase or Fmoc-(AEEA) _x- loaded solid phase with H-(AEEA) (a3) reacting the _solid phase containing free amino groups obtained in step (a1) or (a2) with an N-terminal protected AEEA-containing compound (e.g., Fmoc-AEEA-OH), preferably in the presence of a coupling agent (e.g., carbodiimide or benzotriazole); (a4) draining the solution from the solid phase; (a5) optionally washing the solid phase obtained after step (a4); (a6) optionally repeating steps (a2) to (a5) one or more times to obtain the desired number of repeating units (n) of AEEA; (a7) optionally subjecting the PG-(AEEA) n-loaded solid phase (e.g., Fmoc-(AEEA) n-loaded solid phase) to a final deprotection reaction (e.g., a final Fmoc deprotection reaction when PG is Fmoc), thereby obtaining an H-(AEEA) _n -loaded solid phase; (a8) optionally subjecting the H-(AEEA) _n -loaded solid phase to a final deprotection reaction (e.g., a final Fmoc deprotection reaction when PG is Fmoc), thereby obtaining an H-(AEEA) _n -loaded solid phase; (a9) cleaving the AEEA-polymer obtained in step (a3) _, (a4), (a5), (a6), (a7), or (a8), preferably in the presence of an acid, to obtain _an intermediate compound having formula (VII) or (VII'), wherein for formula (VII), R2 is OH and ^R3 is H, acetyl, or Fmoc; and for formula (VII'), ^R2 is H, acetyl, or Fmoc and ^R3 is OH. In certain embodiments, the intermediate compound having formula (VII) or (VII' ⁾ can be purified with or without pre-purification, for example, using a chromatography system. In certain embodiments, pre-purification includes solvent exchange and/or salt exchange (e.g., by using lyophilization). In certain embodiments, the chromatography system is a liquid chromatography system (e.g., HPLC), wherein the chromatography system preferably includes a suitable detector (e.g., a mass detector). In certain embodiments, after separation using the chromatography system, fractions containing the intermediate compound having Formula (VII) or (VII') (preferably containing the intermediate compound having Formula (VII) or (VII') in sufficient purity (e.g., >90%)) can be collected, pooled, and lyophilized.

In one embodiment of the method for preparing an amphiphilic OEG conjugate compound, particularly when the intermediate compound having formula (VII) or (VII') has an acetylated N-terminus, step (b) comprises the following steps: (b1) reacting Ac-(AEEA) _n -OH with an organic molecule, particularly a compound containing a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety or a monoalkylamine moiety, preferably in the presence of a coupling agent (e.g., a carbodiimide or a benzotriazole), thereby obtaining the Ac-(AEEA) _n -organic molecule, i.e., the amphiphilic OEG conjugate compound having an acetylated N-terminus.

In one embodiment of the method for producing an amphiphilic OEG conjugate compound, particularly when the intermediate compound having formula (VII) or (VII') is N-terminally protected with Fmoc, the compound is subjected to an Fmoc deprotection reaction prior to step (b), thereby obtaining an intermediate compound having formula (VII) or (VII') and a free N-terminus.

In certain embodiments of the method for preparing an amphiphilic OEG conjugate compound, particularly when the intermediate compound having formula (VII) or (VII') has a free N-terminus (i.e., an _NH2 group), step (b) comprises the following steps: (b1') subjecting the N-terminus of H-(AEEA) _n -OH to a protection reaction (e.g., by introducing a BOC protecting group at the N-terminus), thereby obtaining PG-(AEEA) _n -OH (wherein PG is an amino protecting group); (b2') optionally purifying PG-(AEEA) _n -OH (e.g., using a chromatography system); (b3') PG-(AEEA) _n (b4') reacting --OH with an organic molecule, particularly a compound containing a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, a ceramide moiety, or a monoalkylamine moiety, preferably in the presence of a coupling agent (e.g., a carbodiimide or a benzotriazole), thereby obtaining a PG-(AEEA) _n -organic molecule; and (b4') subjecting the PG-(AEEA) _n -organic molecule to a deprotection reaction, thereby obtaining an amphiphilic OEG conjugate compound having a free N-terminus. If desired, the N-terminus can be further derivatized to obtain other amphiphilic OEG conjugate compounds.

In certain embodiments, the amphiphilic OEG conjugate compound can be purified, with or without prior purification, for example, using a chromatography system. In certain embodiments, the prior purification includes solvent exchange and/or salt exchange (e.g., by using lyophilization). In certain embodiments, the chromatography system is a liquid chromatography system (e.g., HPLC), where the chromatography system preferably includes a suitable detector (e.g., a mass detector). In certain embodiments, after separation using the chromatography system, fractions containing the amphiphilic OEG conjugate compound (preferably containing the amphiphilic OEG conjugate compound in sufficient purity (e.g., >90%)) can be collected, pooled, and lyophilized.

The terms " lipid " and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are generally insoluble or poorly soluble in water but are soluble in many organic solvents. In aqueous environments, the amphiphilic nature causes the molecules to self-assemble into organized structures and distinct phases. One of these phases consists of a lipid bilayer, as it exists in aqueous environments as vesicles, multilamellar/unilamellar liposomes, or membranes. Hydrophobicity is conferred by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups may contain polar and/or charged groups, including carbohydrates, phosphate, carboxylic acid, sulfate, amino (e.g., tertiary amino), sulfhydryl, nitro, hydroxyl, and other similar groups.

As used herein, the term "hydrophobic" with respect to a compound, group, or moiety means that the compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, aggregates and excludes water molecules. In certain embodiments, the term "hydrophobic" refers to any compound, group, or moiety that is substantially immiscible or insoluble in an aqueous solution. In certain embodiments, the hydrophobic compound, group, or moiety is substantially non-polar. Examples of hydrophobic groups are hydrocarbyl groups and fluorinated (e.g., perfluorinated) hydrocarbyl groups. In certain embodiments, the hydrophobic compound, group, or moiety is lipophilic. The term hydrophobic group includes hydrocarbons of at least 6 carbon atoms. Monovalent hydrocarbon radicals are referred to herein as hydrocarbyl. Hydrophobic groups can have functional groups (e.g., ethers, esters, halides, etc.) and atoms other than carbon and hydrogen, so long as the group is substantially immiscible or insoluble in an aqueous solution.

As used herein, the term "lipophilic" with respect to a compound, group, or moiety means that the compound, group, or moiety is soluble in a non-polar solvent (e.g., hexane, tetrahydrofuran (THF), and/or chloroform). In certain embodiments, the term "lipophilic" refers to any compound, group, or moiety that is soluble in a non-polar solvent (e.g., hexane, tetrahydrofuran (THF), and/or chloroform) and substantially immiscible or insoluble in aqueous solutions. Examples of lipophilic groups are acyclic, preferably straight-chain, hydrocarbyl groups, e.g., hydrocarbyl groups (e.g., hydrocarbyl groups having at least 10 carbon atoms), such as the lipophilic chains of naturally occurring lipids.

The term "perfluorinated," as used herein with respect to a compound, group, or moiety, means that in the compound, group, or moiety, all C--H moieties have been replaced with C--F moieties. For example, perfluorinated n-octanoic acid has the formula _F3C ( _CF2 ) _6COOH .

The term "hydrocarbon" includes acyclic, e.g., linear (straight-chain) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl, as defined herein. It is recognized that one or more of the hydrogen atoms of an alkyl, alkenyl, or alkynyl may be replaced with other atoms, e.g., halogen, oxygen, or sulfur. Unless otherwise specified, hydrocarbon groups may also include cyclic (alkyl, alkenyl, or alkynyl) groups or aryl groups, so long as the overall polarity of the hydrocarbon remains relatively nonpolar.

As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Often, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion may have a formal positive or negative charge. Alternatively, the polar portion may have both a formal positive and a formal negative charge and may be a zwitterion or inner salt. For purposes of the present invention, an amphiphilic compound may be, but is not limited to, one or more natural or unnatural lipids and lipid-like compounds.

The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances structurally and/or functionally related to lipids but not strictly considered lipids. For example, the term includes compounds capable of forming amphiphilic layers in aqueous environments to reside in vesicles, multilamellar/unilamellar liposomes, or membranes, and includes surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules containing hydrophilic and hydrophobic moieties with various structural organizations that may or may not resemble those of lipids. Examples of lipid-like compounds that can spontaneously incorporate into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSLs), synthetic function-spacer-sterol constructs (FSSs), and artificial amphiphilic molecules. Lipids containing two long alkyl chains and a polar head group are generally cylindrical. The region occupied by the two alkyl chains resembles the region occupied by the polar head group. Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water-insoluble. Traditional surfactant monomers, containing only a single linear alkyl chain and a hydrophilic head group, are generally conical in shape. The hydrophilic head group tends to occupy a larger molecule than the linear alkyl chain. In some embodiments, surfactants tend to aggregate into spherical or ellipsoidal micelles that are water-soluble. While lipids have the same general structure as surfactants—a polar hydrophilic head group and a nonpolar hydrophobic tail—lipids differ from surfactants in the form of their monomers, the type of aggregates they form in solution, and the concentration range required for aggregation. As used herein, the term "lipid" is intended to cover both lipids and lipid-like substances unless otherwise specified herein or clearly contradicted by the context.

Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

In some embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used synonymously with fat, which is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (tri-, di-, and monoglycerides, and phospholipids) as well as steroids, i.e., sterol-containing metabolites such as cholesterol or its derivatives. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

Fatty acids or fatty acid residues are a diverse group of molecules consisting of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that makes it insoluble in water. The carbon chain, typically 4 to 24 carbons long, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is greater the more double bonds in the chain. Other major lipid classes in the fatty acid category are fatty esters and fatty amides.

Glycerolipids consist of mono-, di-, and tri-substituted glycerols, with the most well-known being fatty acid triesters of glycerol called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol by glycosidic bonds.

Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached to two fatty acid-derived "tails" by ester bonds and one "head" group by a phosphate ester bond. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated and have chain lengths of 16 to 26 carbon atoms. The predominant phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules consisting of one or more sugar residues attached via glycosidic bonds to a sphingoid base. Examples of these are simple and complex glycosphingolipids, such as cerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

Glycolipids are compounds in which fatty acids are directly attached to a sugar backbone, forming a structure compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexa-acylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by iterative and multimodular enzymes that share mechanistic properties with classical enzymes as well as fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and exhibit great structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

Cationic or cationically ionizable lipids The nucleic acid (e.g., DNA or RNA) compositions described herein and the nucleic acid particles (particularly RNA LNPs) described herein comprise at least one cationic or cationically ionizable lipid as a particle-forming agent. Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acids. In some embodiments, the cationic or cationically ionizable lipids contemplated for use herein can bind to nucleic acids, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is contained or encapsulated.

As used herein, "cationic lipid" or "cationic lipid-like substance" refers to a lipid or lipid-like substance that has a net positive charge. Cationic lipids or lipid-like substances bind to negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or higher acyl chain, and the lipid head group typically carries a positive charge.

In some embodiments, the cationic lipid or lipid-like substance has a net positive charge only at certain pHs, particularly acidic pHs, while preferably having no net charge, i.e., neutral, at a different, preferably higher, pH, such as physiological pH. This ionizable behavior is believed to enhance efficacy and reduce toxicity by aiding in endosomal escape, compared to particles that remain cationic at physiological pH.

As used herein, "cationically ionizable lipid" refers to a lipid or lipid-like substance that has a net positive charge or is neutral, i.e., a lipid that is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is dissolved, the cationically ionizable lipid is positively charged or neutral. For purposes of the present invention, such "cationically ionizable" lipids are included within the term "cationic lipid," unless the context requires otherwise.

Examples of cationic lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-methylammoniumpropane; -dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradeoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE) and 2,3-dioleoyloxy -N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy) (cis)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP) ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanamin N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy) -1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1- [2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di((Z)non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)-dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)-oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethyl-amino)propionamide (Lipidoid 98N ₁₂ -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200) and the following structures (XV-1) to (XV-6):
Including, but not limited to:

DODMA, DOTMA, DOTAP, DODAC, and DOSPA are preferred. In a specific embodiment, the cationic or cationically ionizable lipid is DODMA.

DOTMA is a cationic lipid with a quaternary amine head group. The structure of DOTMA can be represented as follows:

DODMA is an ionizable cationic lipid with a tertiary amine head group. The structure of DODMA can be represented as follows:

In some embodiments, the composition includes a cationically ionizable lipid.

In some embodiments, the cationically ionizable lipid comprises a head group that includes at least one nitrogen atom (N) that is capable of being positively charged or protonated, preferably under physiological conditions. In some embodiments, the cationically ionizable lipid comprises a head group that includes at least one tertiary amine moiety.

Examples of cationically ionizable lipids are disclosed, for example, in WO 2016/176330 and WO 2018/078053. In some embodiments, the cationically ionizable lipid has the formula (X):
[During the ceremony,
One of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)—, —C(═O)NR ^a —, NR ^a C(═O)NR ^a —, —OC(═O)NR ^a — or —NR ^a C(═O)O—; and the other of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)NR ^a -, NR ^a C(═O)NR ^a -, -OC(═O)NR ^a - or -NR ^a C(═O)O- or a direct bond;
G ¹ and G ² are each independently unsubstituted C ₁ -C ₁₂ alkylene or C _2-12 alkenylene;
^G3 is _C1-24 alkylene, _C2-24 alkenylene, _C3-8 cycloalkylene or _C3-8 cycloalkenylene;
R ^a is H or C _1-12 alkyl;
R ³⁵ and R ³⁶ are each independently C _6-24 alkyl or C _6-24 alkenyl;
R ³⁷ is H, OR ⁵⁰ , CN, —C(═O)OR ⁴⁰ , —OC(═O)R ⁴⁰ or —NR ⁵⁰ C(═O)R ⁴⁰ ;
R ⁴⁰ is C _1-12 alkyl;
R ⁵⁰ is H or C _1-6 alkyl; and x is 0, 1, or 2.
or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

In some of the above embodiments of formula (X), the lipid has the following structure (XA) or (XB):
[In the ceremony:
A is a 3- to 8-membered cycloalkyl or cycloalkylene group;
R ⁶⁰ , at each occurrence, is independently H, OH, or C ₁ -C ₂₄ alkyl;
n1 is an integer ranging from 1 to 15.
It has one of the following.

In some of the above embodiments of formula (X), the lipid has structure (XA), and in other embodiments, the lipid has structure (XB).

In other embodiments of formula (X), the lipid has the following structure (XC) or (XD):
(wherein y and z are each independently an integer ranging from 1 to 12.)
It has one of the following.

In any of the above embodiments of Formula (X), one of L ¹⁰ and L ²⁰ is -O(C=O)-. For example, in certain embodiments, each of L ¹⁰ and L ²⁰ is -O(C=O)-. In certain different embodiments of any of the above, L ¹⁰ and L ²⁰ are each independently -(C=O)O- or -O(C=O)-. For example, in certain embodiments, each of L ¹⁰ and L ²⁰ is -(C=O)O-.

In certain different embodiments of formula (X), the lipid has the following structure (XE) or (XF):
It has one of the following.

In some of the above embodiments of formula (X), the lipid has the following structure (XG), (XH), (XJ) or (XK):
It has one of the following.

In some of the above embodiments of Formula (X), n1 is an integer ranging from 2 to 12, e.g., from 2 to 8 or from 2 to 4. For example, in some embodiments, n1 is 3, 4, 5, or 6. In some embodiments, n1 is 3. In some embodiments, n1 is 4. In some embodiments, n1 is 5. In some embodiments, n1 is 6.

In certain other embodiments of Formula (X), y and z are each independently an integer ranging from 2 to 10. For example, in certain embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

In some of the above embodiments of Formula (X), R ⁶⁰ is H. In others of the above embodiments, R ⁶⁰ is C ₁ -C ₂₄ alkyl. In other embodiments, R ⁶⁰ is OH.

In some embodiments of Formula (X), ^G3 is unsubstituted. In other embodiments, ^G3 is substituted. In various different embodiments, ^G3 is a straight chain _C1 - _C24 alkylene or a straight chain _C2 - _C24 alkenylene.

In certain other such embodiments of Formula (X), R ³⁵ or R ³⁶ or both are C ₆ -C ₂₄ alkenyl. For example, in certain embodiments, R ³⁵ and R ³⁶ each independently have the following structure:
[In the ceremony:
R ^7a and R ^7b , at each occurrence, are independently H or C ₁ -C ₁₂ alkyl; and a is an integer from 2 to 12;
wherein R ^7a , R ^7b and a are selected such that R ³⁵ and R ³⁶ each independently contain 6 to 20 carbon atoms.
It has.
For example, in certain embodiments, a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the above embodiments of Formula (X), at least one of R ^7a is H. For example, in certain embodiments, R ^7a is H at each occurrence. In other different embodiments of the above, at least one of R ^7b is C ₁ -C ₈ alkyl. For example, in certain embodiments, C ₁ -C ₈ alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

In different embodiments of formula (X), R ³⁵ or R ³⁶ or both have the following structure:
It has.

In some of the above embodiments of Formula (X), R ³⁷ is OH, CN, —C(═O)OR ⁴⁰ , —OC(═O)R ⁴⁰ , or —NHC(═O)R ^40. In certain embodiments, R ⁴⁰ is methyl or ethyl.

In various different embodiments, the cationically ionizable lipid of formula (X) has one of the structures shown below:

In various different embodiments, the cationically ionizable lipid has one of the structures shown in the table below.

In some embodiments, the cationically ionizable lipid has formula (XI):
[During the ceremony,
each of R ₁ and R ₂ is independently R ₅ or -G ₁ -L ₁ -R ₆ , where at least one of R ₁ and R ₂ is -G ₁ -L ₁ -R ₆ ;
Each of R ₃ and R ₄ is independently selected from the group consisting of C _1-6 alkyl, C _2-6 alkenyl, aryl, and C _3-10 cycloalkyl;
Each of _R5 and _R6 is independently an acyclic hydrocarbyl group having at least 10 carbon atoms;
each of G ₁ and G ₂ is independently unsubstituted C _1-12 alkylene or C _2-12 alkenylene;
each of L ₁ and L ₂ is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, —NRaC(═O)NRa—, —OC(═O)NRa— and —NRaC(═O)O—;
Ra is H or C _1-12 alkyl;
m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2.
It has the following structure.

In some of the above embodiments of Formula (XI), G ₁ is independently unsubstituted C ₁ -C ₁₂ alkylene or unsubstituted C _2-12 alkenylene, for example, unsubstituted, straight-chain C _1-12 alkylene or unsubstituted, straight-chain C _2-12 alkenylene. In some embodiments, each G ₁ is independently unsubstituted C _6-12 alkylene or unsubstituted C _6-12 alkenylene, for example, unsubstituted, straight-chain C _6-12 alkylene or unsubstituted, straight-chain C _6-12 alkenylene. In some embodiments, each G ₁ is independently unsubstituted C _8-12 alkylene or unsubstituted C _8-12 alkenylene, for example, unsubstituted, straight-chain C _8-12 alkylene or unsubstituted, straight-chain C _8-12 alkenylene. In certain embodiments, each G ₁ is independently unsubstituted C _6-10 alkylene or unsubstituted C _6-10 alkenylene, for example, unsubstituted, straight-chain C _6-10 alkylene or unsubstituted, straight-chain C _6-10 alkenylene. In certain embodiments, each G ₁ is independently unsubstituted alkylene having 8, 9, or 10 carbon atoms, for example, unsubstituted, straight-chain alkylene having 8, 9, or 10 carbon atoms. In certain embodiments, when both R ₁ and R ₂ are independently -G ₁ -L ₁ -R ₆ , G ₁ of R ₁ can be different from G ₁ of R ₂ . In some of these embodiments, for example, G ₁ of R ₁ is an unsubstituted, linear C _1-12 alkylene and G ₁ of R ₂ is an unsubstituted, linear C _2-12 alkenylene; or G ₁ of R ₁ is an unsubstituted, linear C _1-12 alkylene group and G ₁ of R ₂ is a different unsubstituted, linear C _1-12 alkylene group. In certain embodiments, when both R ₁ and R ₂ are independently -G ₁ -L ₁ -R ₆ , G ₁ of R ₁ can be the same as G ₁ of R _2. In some of these embodiments, for example, each G ₁ is the same unsubstituted, linear C _8-12 alkylene, e.g., unsubstituted, linear C _8-10 alkylene, or each G ₁ is the same unsubstituted, linear C _6-12 alkenylene.

In some of the above embodiments of Formula (XI), each L ₁ is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, and —C(═O)NRa-. In some embodiments, Ra of L ₁ is H or C _1-12 alkyl. In some embodiments, Ra of L ₁ is H or C _1-6 alkyl, e.g., H or C _1-3 alkyl. In some embodiments, Ra of L ₁ is H, methyl, or ethyl. In some embodiments, each L ₁ is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)S—, and —SC(═O)—. In some embodiments, each L ₁ is independently —O(C═O)— or —(C═O)O—. In certain embodiments, when both R ₁ and R ₂ are independently -G ₁ -L ₁ -R ₆ , L ₁ of R ₁ can be different from L ₁ of R _2. In some of these embodiments, for example, L ₁ of R ₁ is a moiety selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, and —C(═O)NRa- (e.g., L ₁ of R ₁ is —O(C═O)—), and L ₁ of R ₂ is a different moiety selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, and —C(═O)NRa- (e.g., L ₁ of R ₂ is —(C═O)O—). In certain embodiments, when both R ₁ and R ₂ are independently -G ₁ -L ₁ -R ₆ , L ₁ of R ₁ can be the same as L ₁ of R _2. In some of these embodiments, for example, each L ₁ is the same moiety selected from the group consisting of -O(C═O)-, -(C═O)O-, -C(═O)S-, -SC(═O)-, -NRaC(═O)-, and -C(═O)NRa-, e.g., each L ₁ is -O(C═O)- or each L ₁ is -(C═O)O-.

In some of the above embodiments of Formula (XI), each _R6 is independently an acyclic hydrocarbyl group having at least 10 carbon atoms, e.g., a linear hydrocarbyl group having at least 10 carbon atoms. In certain embodiments, each _R6 independently has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments, each _R6 is independently an acyclic hydrocarbyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a linear hydrocarbyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In certain embodiments, each _R6 is bonded to _L1 via an internal carbon atom of _R6 . In certain embodiments, each _R6 independently has up to 30 carbon atoms (e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms), and each _R6 is bonded to _L1 via an internal carbon atom of _R6 . In certain embodiments, each _R6 is independently an acyclic hydrocarbyl group having at least 10 carbon atoms, for example, a linear hydrocarbyl group having at least 10 carbon atoms, and each _R6 is bonded to _L1 via an internal carbon atom of _R6 . In certain embodiments, each _R6 is independently an acyclic hydrocarbyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), e.g., a straight-chain hydrocarbyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each _R6 is bonded to _L1 through an internal carbon atom of _R6 . In certain embodiments, the hydrocarbyl group of _R6 is an alkyl or alkenyl group, e.g., a _C10-30 alkyl or alkenyl group. Thus, in certain embodiments, each _R6 is independently an acyclic alkyl group having at least 10 carbon atoms or an acyclic alkenyl group having at least 10 carbon atoms, e.g., a straight-chain alkyl group having at least 10 carbon atoms or a straight-chain alkenyl group having at least 10 carbon atoms. In certain embodiments, each _R6 is independently an acyclic alkyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or an acyclic alkenyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a straight-chain alkyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a straight-chain alkenyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments, each _R6 is independently an acyclic alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), e.g., a linear alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms). In certain embodiments, each _R6 is independently an acyclic alkyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or an acyclic alkenyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear alkyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms) or a linear alkenyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and each _R6 is bonded to _L1 via an internal carbon atom of _R6 . In certain embodiments, each _R6 is independently an acyclic alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), e.g., a straight-chain alkyl group having 11 to 19 carbon atoms (e.g., 11, 13, 15, 17, or 17 carbon atoms), and each _R6 is bonded to _L1 via an internal carbon atom of _R6 . The expression "internal carbon atom" means that the carbon atom of _R6 at which _R6 is bonded to _L1 is directly bonded to at least two other carbon atoms of _R6 . For example, with respect to the following _C11 alkyl group, any of the carbon atoms at positions 2, 3, 4, 5, and 7 qualify as an "internal carbon atom" according to the present invention, while the carbon atoms at positions 1, 6, 8, 9, 10, and 11 do not.

As a result, _R6 , a _C11 alkyl group attached to _L1 through an internal carbon atom of _R6 , is selected from the group:
[During the ceremony,
represents the bond connecting _R6 to _L1 . Furthermore, for a straight-chain alkyl group, e.g., a straight-chain _C11 alkyl group, each carbon atom other than the first and last carbon atom of the straight-chain alkyl group (i.e., other than the carbon atoms at positions 1 and 11) qualifies as an "internal carbon atom." Thus, in certain embodiments, _R6 being a straight-chain alkyl group having p carbon atoms and connecting to _L1 via an internal carbon atom of _R6 means that _R6 is connected to _L1 at any carbon _atom from positions 2 to (p-1) of R6 (thereby excluding the terminal C atoms at positions 1 and p). In one embodiment, when _R6 is a straight-chain alkyl group having p' carbon atoms (where p' is an even number) and bonded to _L1 via an internal carbon atom of _R6 , _R6 bonds to _L1 via _any of the carbon atoms in the (p'/2-1), (p'/2) and (p'/2+1) positions of R6 ₍ e.g., if p' is 10, _R6 bonds to _L1 via any of the carbon atoms in the 4th, 5th and 6th positions of R6). In certain embodiments, when _R6 is a straight-chain alkyl group having p" carbon atoms (where p" is an odd number) and bonded to _L1 via an internal carbon atom of _R6 , _R6 bonds to _L1 via either _the (p"-1)/2 or (p"+1)/2 carbon atom of R6 (e.g., if p" is 11, _R6 bonds to _L1 via either the 5 or 6 carbon atom of _R6 ). In general, if both _R1 and _R2 are _-G1 - _L1 - _R6 and each _R6 is bonded to _L1 through an internal carbon atom of _R6 , it is understood that _R6 of _R1 is bonded to _L1 of _R1 (and not _L1 of _R2 ) through an internal carbon atom of _R6 of R1, _{and R6 of R2} is bonded to _L1 of _R2 (and not _L1 of _R1 ) through an internal carbon atom of _R6 of _R2 . In certain embodiments, each _R6 is:
wherein:
represents the bond connecting _R6 to _L1 . In certain embodiments, when both _R1 and _R2 are independently _-G1 - _L1 - _R6 , _R6 of _R1 is different from _R6 of _R2 . In some of these embodiments, for example, _R6 of _R1 can be an acyclic, preferably linear, hydrocarbyl group having at least 10 carbon atoms (e.g., _R6 of _R1 is
R ₂ and R ₆ may be different acyclic, preferably linear, hydrocarbyl groups having at least 10 carbon atoms (e.g., R ₂ and R ₆ may be
In certain embodiments, when both R ₁ and R ₂ are independently -G ₁ -L ₁ -R ₆ , R ₆ of R ₁ is the same as R ₆ of R _2. In some of these embodiments, for example, each R ₆ is the same acyclic, preferably linear, hydrocarbyl group having at least 10 carbon atoms (e.g., each R ₆ is
(It is).

In some of the above embodiments of Formula (XI), _R5 is an acyclic hydrocarbyl group having at least 10 carbon atoms, e.g., a linear hydrocarbyl group having at least 10 carbon atoms. In certain embodiments, _R5 is an acyclic hydrocarbyl group having at least 12 carbon atoms, e.g., at least 14, at least 16, or at least 18 carbon atoms, e.g., a linear hydrocarbyl group having at least 12, at least 14, at least 16, or at least 18 carbon atoms. In certain embodiments, _R5 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments, R ₅ is an acyclic hydrocarbyl group, for example, a linear hydrocarbyl group, where each hydrocarbyl group has from 10 to 30 carbon atoms (e.g., from 10 to 28, from 10 to 26, from 10 to 24, from 10 to 22, from 10 to 20 carbon atoms or from 12 to 30, from 12 to 28, from 12 to 26, from 12 to 24, from 12 to 22, from 12 to 20 carbon atoms or In certain embodiments, the hydrocarbyl group of R5 is an alkyl or alkenyl group, for example, a _{C10-30 alkyl} or alkenyl group. Thus, in certain embodiments, _R5 is an acyclic alkyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms) or an acyclic alkenyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms), such as a linear alkyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms) or a linear alkenyl group having at least 10 carbon atoms (e.g., at least 12, at least 14, at least 16, or at least 18 carbon atoms). ₅ is a non-cyclic alkyl group or a non-cyclic alkenyl group, e.g., a straight chain alkyl group or a straight chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, In some embodiments, the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds, e.g., two carbon-carbon double bonds. In some embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., one, two, or three, e.g., two carbon-carbon double bonds in the cis configuration. Thus, in some embodiments, R ₅ is a non-cyclic alkyl group or a non-cyclic alkenyl group, for example, a straight chain alkyl group or a straight chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms or 14 to 30, 14 to 2 In certain embodiments, R has 16-30, 16-28, 16-26, 16-24, 16-22, 16-20 carbon atoms or 18-30, 18-28, 18-26, 18-24, 18-22, or 18-20 carbon atoms), and the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds. ₅ is a non-cyclic alkyl group or a non-cyclic alkenyl group, for example, a straight chain alkyl group or a straight chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20 carbon atoms or 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22, 12 to 20 carbon atoms or 14 to 30, 14 to 28 ... In some embodiments, R ₅ has the following structure:
where
represents the bond where ^R5 is attached to the rest of the compound.

In some of the above embodiments of Formula (XI), _L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, and -NRaC(=O)O-. In certain embodiments, _L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In certain embodiments, Ra of _L2 is H or C _1-12 alkyl. In certain embodiments, Ra of _L2 is H or C _1-6 alkyl, for example, H or C _1-3 alkyl. In certain embodiments, Ra of _L2 is H, methyl, or ethyl. In certain embodiments, _L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, and -SC(=O)-. In certain embodiments, _L2 is -O(C=O)- or -(C=O)O-.

In some of the above embodiments of Formula (XI), _G2 is unsubstituted _C1-12 alkylene or unsubstituted _C2-12 alkenylene, for example, unsubstituted, straight-chain _C1-12 alkylene or unsubstituted, straight-chain _C2-12 alkenylene. In some embodiments, _G2 is unsubstituted _C2-10 alkylene or unsubstituted _C2-10 alkenylene, for example, unsubstituted, straight-chain _C2-10 alkylene or unsubstituted, straight-chain _C2-10 alkenylene. In some embodiments, _G2 is unsubstituted _C2-6 alkylene or unsubstituted _C2-6 alkenylene, for example, unsubstituted, straight-chain _C2-6 alkylene or unsubstituted, straight-chain _C2-6 alkenylene. In certain embodiments, _G2 is unsubstituted _C2-4 alkylene or unsubstituted _C2-4 alkenylene, for example, unsubstituted, straight-chain _C2-4 alkylene or unsubstituted, straight-chain _C2-4 alkenylene. In certain embodiments, _G2 is ethylene or trimethylene.

In some of the above embodiments of Formula (XI), each of _R3 and _R4 is independently _C1-6 alkyl or _C2-6 alkenyl. In certain embodiments, each of _R3 and _R4 is independently _C1-4 alkyl or _C2-4 alkenyl. In certain embodiments, each of _R3 and _R4 is independently _C1-3 alkyl. In certain embodiments, each of _R3 and _R4 is independently methyl or ethyl. In certain embodiments, each of _R3 and _R4 is methyl.

In some of the above embodiments of Formula (XI), m is 0, 1, 2, or 3. In some embodiments, m is 0 or 2. In some embodiments, m is 0. In some embodiments, m is 2.

In some of the above embodiments of Formula (XI), the cationically ionizable lipid has Formula (XIIa) or (XIIb):
[During the ceremony,
Each of R ₃ and R ₄ is independently C ₁ -C ₆ alkyl or C _2-6 alkenyl;
_R5 is a linear hydrocarbyl group having at least 14 carbon atoms (e.g., at least 16 carbon atoms), where the hydrocarbyl group preferably has at least two carbon-carbon double bonds;
Each R ₆ is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms and/or each R ₆ is bonded to L ₁ through an internal carbon atom of R ₆ , preferably each R ₆ is independently a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms and each R ₆ is bonded to L ₁ through an internal carbon atom of R ₆ ;
each _G1 is independently an unsubstituted, straight-chain _C4-12 alkylene or _C4-12 alkenylene, for example, an unsubstituted, straight-chain _C6-12 alkylene or _C6-12 alkenylene, for example, an unsubstituted, straight-chain _C8-12 alkylene or an unsubstituted, straight-chain _C8-12 alkenylene;
_G2 is unsubstituted _C2 - _C10 alkylene or _C2-10 alkenylene, preferably unsubstituted _C2 - _C6 alkylene or _C2-6 alkenylene;
Each of _L1 and _L2 is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2, or 3, preferably 0 or 2.
It has the following structure.

In some of the above embodiments of Formula (XIIa), _R5 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments of Formula (XIIa), _R5 is a linear hydrocarbyl group having 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In certain embodiments of Formula (XIIa), _R5 is a straight-chain alkyl or alkenyl group having 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In certain embodiments of Formula (XIIa), the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds, e.g., two carbon-carbon double bonds. In certain embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, eg, 1, 2 or 3, eg, 2, carbon-carbon double bonds in the cis configuration. Thus, in certain embodiments of Formula (XIIa), _R5 is a straight chain alkyl group or a straight chain alkenyl group, wherein the alkyl and alkenyl groups each independently have 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds. In certain embodiments of Formula (XIIa), _R5 is a straight-chain alkyl group or a straight-chain alkenyl group, wherein the alkyl and alkenyl groups each independently have 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., 1, 2, or 3 carbon-carbon double bonds. In certain embodiments of Formula (XIIa), _R5 is a straight chain alkyl group or a straight chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 14 to 30 carbon atoms (e.g., 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20 carbon atoms, or 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms, or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has two or three carbon-carbon double bonds, wherein at least one carbon-carbon double bond, e.g., one, two, or three carbon-carbon double bonds, is in the cis configuration. In certain embodiments of Formula (XIIa), ^R5 has the following structure:
where
represents the bond through which ^R5 is attached to the remainder of the compound. In some embodiments of Formula (XIIa), _R6 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In some embodiments of Formula (XIIa), _R6 is an acyclic hydrocarbyl group (e.g., an acyclic alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms). In some embodiments of Formula (XIIa), _R6 is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms, and ^R6 is attached to _L1 through an internal carbon atom of _R6 . In some embodiments of Formula (XIIa), _R6 is an acyclic hydrocarbyl group (e.g., a linear alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), for example, a linear hydrocarbyl group (e.g., a linear alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and _R6 is attached to _L1 through an internal carbon atom of _R6 . In some embodiments of Formula (XIIa), _G1 is independently unsubstituted, linear _C4-12 alkylene or _C4-12 alkenylene, for example, unsubstituted, linear _C6-12 alkylene or _C6-12 alkenylene. In some embodiments of Formula (XIIa), _R5 is a linear hydrocarbyl group having at least 14 carbon atoms (e.g., 14 to 30 carbon atoms) and two or three carbon-carbon double bonds, for example, a linear alkenyl group; _R6 is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms (e.g., 10 to 30 carbon atoms), and _R6 is bonded to _L1 via an internal carbon atom of _R6 ; and _G1 is independently unsubstituted, linear _C4-12 alkylene or _C4-12 alkenylene, for example, unsubstituted, linear _C6-12 alkylene or _C6-12 alkenylene.

In some of the above embodiments of Formula (XIIb), each _R6 independently has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments of Formula (XIIb), each _R6 independently is a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms). In certain embodiments of Formula (XIIb), each _R6 is independently a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms), and each _R6 is bonded to _L1 through an internal carbon atom of _R6 . In certain embodiments of Formula (XIIb), each _R6 is independently:
wherein:
represents the bond connecting _R6 to _L1 . In certain embodiments of Formula (XIIb), each _G1 is independently unsubstituted, straight chain _C6-12 alkylene or _C6-12 alkenylene. In certain embodiments of Formula (XIIb), each _G1 is independently unsubstituted, straight chain _C8-12 alkylene or _C8-12 alkenylene. In certain embodiments of Formula (XIIb), each _R6 is independently a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having at least 10 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms) and is attached to _L1 through an internal carbon atom of _R6 ; and each _G1 is independently unsubstituted, straight-chain _C8-12 alkylene or _C8-12 alkenylene.

In some of the above embodiments of Formula (XI), the cationically ionizable lipid has Formula (XIIIa) or (XIIIb):
[During the ceremony,
each of R ₃ and R ₄ independently is C _1-4 alkyl or C _2-4 alkenyl, more preferably C _1-3 alkyl, for example methyl or ethyl;
_R5 is a straight chain alkyl or alkenyl group having at least 16 carbon atoms, where the alkenyl group preferably has at least two carbon-carbon double bonds;
each _R6 is independently a linear hydrocarbyl group having at least 10 carbon atoms, wherein _R6 is attached to _L1 through an internal carbon atom of _R6 ;
each _G1 is independently unsubstituted, straight-chain _C6-12 alkylene or unsubstituted, straight-chain _C6-12 alkenylene, for example, unsubstituted, straight-chain _C8-12 alkylene or unsubstituted, straight-chain _C8-12 alkenylene, for example, unsubstituted, straight-chain _C8-10 alkylene or unsubstituted, straight-chain _C8-10 alkenylene, for example, unsubstituted, straight-chain _C8 alkylene;
_G2 is unsubstituted _C2-6 alkylene or _C2-6 alkenylene, preferably unsubstituted _C2-4 alkylene or _C2-4 alkenylene, for example ethylene or trimethylene;
Each of _L1 and _L2 is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2, or 3, preferably 0 or 2.
It has the following structure.

In some of the above embodiments of Formula (XIIIa), _R5 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments of Formula (XIIIa), _R5 is a straight-chain alkyl or alkenyl group having 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms). In certain embodiments of Formula (XIIIa), the alkenyl group has at least two carbon-carbon double bonds, e.g., two or three carbon-carbon double bonds, e.g., two carbon-carbon double bonds. In certain embodiments, the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., 1, 2, or 3, e.g., 2, carbon-carbon double bonds in the cis configuration. Thus, in certain embodiments of Formula (XIIIa), _R5 is a straight chain alkyl group or a straight chain alkenyl group, wherein the alkyl and alkenyl groups each independently have 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least two carbon-carbon double bonds, e.g., 2 or 3 carbon-carbon double bonds. In certain embodiments of Formula (XIIIa), _R5 is a straight-chain alkyl group or a straight-chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has at least one carbon-carbon double bond in the cis configuration, e.g., 1, 2, or 3 carbon-carbon double bonds. In certain embodiments of Formula (XIIIa), _R5 is a straight chain alkyl group or a straight chain alkenyl group, wherein each of the alkyl and alkenyl groups independently has 16 to 30 carbon atoms (e.g., 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20 carbon atoms or 18 to 30, 18 to 28, 18 to 26, 18 to 24, 18 to 22, or 18 to 20 carbon atoms), and the alkenyl group has two or three carbon-carbon double bonds, wherein at least one carbon-carbon double bond, e.g., one, two, or three carbon-carbon double bonds, is in the cis configuration. In certain embodiments of Formula (XIIIa), _R5 has the following structure:
where
represents the bond through which ^R5 is attached to the remainder of the compound. In certain embodiments of Formula (XIIIa), _R6 has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments of Formula (XIIIa), _R6 is a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and _R6 is attached to _L1 through an internal carbon atom of _R6 . In certain embodiments of Formula (XIIIa), _R6 is a straight-chain alkyl group having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms), and _R6 is attached to _L1 through an internal carbon atom of _R6 . In some embodiments of Formula (XIIIa), _G1 is independently unsubstituted, linear _C4-12 alkylene or _C4-12 alkenylene, for example, unsubstituted, linear _C6-12 alkylene or _C6-12 alkenylene. In some embodiments of Formula (XIIIa), _R5 is a linear hydrocarbyl group having at least 16 carbon atoms (e.g., 16 to 30 carbon atoms) and two or three carbon-carbon double bonds, for example, a linear alkenyl group; _R6 is a linear hydrocarbyl group (e.g., a linear alkyl group) having at least 10 carbon atoms (e.g., 10 to 30 carbon atoms), and _R6 is bonded to _L1 via an internal carbon atom of _R6 ; and _G1 is independently unsubstituted, linear _C4-12 alkylene or _C4-12 alkenylene, for example, unsubstituted, linear _C6-12 alkylene or _C6-12 alkenylene.

In some of the above embodiments of Formula (XIIIb), each _R6 independently has up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, or up to 20 carbon atoms. In certain embodiments of Formula (XIIIb), each _R6 independently is a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having 10 to 30 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms), and each _R6 is bonded to _L1 through an internal carbon atom of _R6 . In certain embodiments of Formula (XIIIb), each _R6 is bonded to _L1 through an internal carbon atom of _R6 and is independently:
wherein:
represents the bond connecting R ₆ to L _1. In certain embodiments of Formula (XIIIb), each G ₁ is independently an unsubstituted, straight-chain C _8-12 alkylene or C _8-12 alkenylene, for example, an unsubstituted, straight-chain C _8-10 alkylene or C _8-10 alkenylene. In certain embodiments of Formula (XIIIb), each _R6 is independently a straight-chain hydrocarbyl group (e.g., a straight-chain alkyl group) having at least 10 carbon atoms (e.g., 10 to 28, 10 to 26, 10 to 24, 10 to 22, or 10 to 20 carbon atoms or 11 to 19 carbon atoms, e.g., 11, 13, 15, 17, or 17 carbon atoms) and is attached to _L1 through an internal carbon atom of _R6 ; and each _G1 is independently an unsubstituted, straight-chain _C8-12 alkylene or _C8-12 alkenylene, e.g., an unsubstituted, straight-chain _C8-10 alkylene or _C8-10 alkenylene.

In some of the above embodiments of formula (XI), the cationically ionizable lipid has the following formulas (XIV-1), (XIV-2), and (XIV-3):
It has one of the following.

In some embodiments, the cationically ionizable lipid is (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate (3D-P-DMA). The structure of 3D-P-DMA can be represented as follows:

In various different embodiments, the cationically ionizable lipid is selected from the group consisting of N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), and 4-((di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)-N,N-dimethyl-4-oxobutan-1-amine (DPL-14).

Further examples of cationically ionizable lipids include 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane, 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine ( DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP) , 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), Heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-M C3-DMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), di((Z)non-2-en-1-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{ These include, but are not limited to, 2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid C12-200).

In some embodiments, the cationically ionizable lipid has the structure X-3.

In some embodiments, the cationic lipid used herein is or comprises DPL-14. As used herein, "DPL-14" refers to a lipid comprising the general formula:

It is understood that any reference to a cationically ionizable lipid disclosed herein also includes its salts (particularly pharmaceutically acceptable salts), tautomers, stereoisomers, solvates (e.g., hydrates), and isotopically labeled forms.

In some embodiments, the cationically ionizable lipids may comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipid/particles present in the composition. In some embodiments, the cationically ionizable lipids comprise about 40 mol% to about 75 mol%, preferably about 40 mol% to about 70 mol%, and more preferably about 45 mol% to about 65 mol% of the total lipid/particles present in the composition.

In some embodiments where the nucleic acid compositions/particles (particularly RNA compositions/particles) described herein comprise a cationically ionizable lipid and one or more additional lipids, the cationically ionizable lipid comprises about 10 mol% to about 80 mol%, about 20 mol% to about 75 mol%, about 20 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 40 mol% to about 55 mol% of the total lipid/particle present in the composition.

In some embodiments of the nucleic acid (e.g., DNA or RNA) compositions (particularly mRNA compositions) described herein, when at least a portion of (i) the nucleic acid and (ii) the cationic or cationically ionizable lipids form a particle (e.g., an LNP), the cationic or cationically ionizable lipids may comprise about 10 mol% to about 80 mol%, about 20 mol% to about 75 mol%, about 20 mol% to about 70 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 40 mol% to about 55 mol% of the total lipids present in the particle.

In some embodiments, the N/P value is at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

Additional Lipids In some embodiments, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles) described herein can also contain lipids or lipid-like substances other than cationic or cationically ionizable lipids, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances). Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances. In some embodiments, optimizing the formulation of nucleic acid particles by adding other hydrophobic moieties, such as cholesterol and lipids, in addition to cationically ionizable lipids can enhance composition and/or particle stability and the efficacy of nucleic acid (e.g., DNA or RNA) delivery.

One or more additional lipids are incorporated, which may or may not affect the overall charge of the nucleic acid particle. In some embodiments, the one or more additional lipids are non-cationic lipids or lipid-like substances. Non-cationic lipids can include, for example, one or more anionic lipids and/or neutral lipids. As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. As used herein, "neutral lipid" refers to any of several lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions/particles (particularly mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and one or more additional lipids.

While not wishing to be bound by theory, the amount of cationic/cationically ionizable lipid relative to the amount of one or more additional lipids can affect important nucleic acid particle characteristics, such as nucleic acid charge, particle size, stability, tissue selectivity, and bioactivity. Thus, in some embodiments, the molar ratio of cationic/cationically ionizable lipid to one or more additional lipids is from about 10:0 to about 1:9, from about 4:1 to about 1:2, or from about 3:1 to about 1:1.

In some embodiments, the one or more additional lipids included in the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein include one or more of the following: neutral lipids, steroids, and combinations thereof. In some embodiments, the one or more additional lipids included in the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein include a combination of a neutral lipid and a steroid.

Neutral lipids In some embodiments, the one or more additional lipids comprise a neutral lipid, preferably a phospholipid. In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and sphingomyelin. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. Such phospholipids are in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM), and additional phosphatidylethanolamine lipids with various hydrophobic chains. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments, the neutral lipid is DSPC.

Thus, in some embodiments, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and a phospholipid. In some embodiments, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and a phospholipid selected from the group consisting of DSPC, DPPC, DSPE, and DPPE. In some embodiments, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and DSPC.

In some embodiments, neutral lipids are present in a nucleic acid composition (e.g., a DNA or RNA composition, particularly an mRNA composition) described herein at a concentration ranging from about 5 mol% to about 40 mol%, e.g., from about 5 mol% to about 25 mol%, from about 5 mol% to about 20 mol%, from about 5 mol% to about 15 mol%, or from about 5 mol% to about 10 mol% of the total lipids present in the nucleic acid composition (e.g., a DNA or RNA composition, particularly an mRNA composition) described herein.

In some embodiments of the nucleic acid (e.g., DNA or RNA) compositions (particularly mRNA compositions) described herein, when a portion of (i) the nucleic acid (e.g., RNA), (ii) the cationic/cationically ionizable lipid, and (iii) the neutral lipids form a particle (e.g., LNP), the neutral lipids (e.g., one or more phospholipids) can comprise about 5 mol% to about 40 mol%, e.g., about 5 mol% to about 25 mol%, about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, or about 5 mol% to about 10 mol% of the total lipids present in the particle.

In some embodiments, the steroid is cholesterol. Thus, in some embodiments, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise a cationic/cationically ionizable lipid and cholesterol.

In some embodiments, the steroid is present in a nucleic acid composition (e.g., a DNA or RNA composition, particularly an mRNA composition) described herein at a concentration ranging from about 10 mol% to about 65 mol%, e.g., about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol%, or about 25 mol% to about 35 mol% of the total lipids present in the composition (e.g., an mRNA composition) described herein.

In some embodiments of the nucleic acid (e.g., DNA or RNA) compositions (particularly mRNA compositions) described herein, when the nucleic acid (e.g., RNA), (ii) cationic/cationically ionizable lipid, and (iii) steroid (e.g., cholesterol) form a particle (e.g., LNP), the steroid may comprise about 10 mol% to about 65 mol%, e.g., about 20 mol% to about 60 mol%, about 30 mol% to about 50 mol%, or about 25 mol% to about 35 mol% of the total lipid present in the particle.

In a preferred embodiment, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise phospholipids and cholesterol, preferably at the concentrations described above. In a preferred embodiment, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise phospholipids and cholesterol selected from the group consisting of DSPC, DPPC, DSPE, and DPPE, preferably at the concentrations described above. In a preferred embodiment, the nucleic acid compositions/particles (e.g., DNA or RNA compositions/particles, particularly mRNA compositions/particles) described herein comprise DSPC and cholesterol, preferably at the concentrations described above.

In some embodiments, the combined concentration of neutral lipids (particularly, one or more phospholipids) and steroids (particularly, cholesterol) can comprise from about 0 mol% to about 70 mol%, e.g., from about 2 mol% to about 60 mol%, from about 5 mol% to about 55 mol%, from about 5 mol% to about 50 mol%, from about 5 mol% to about 40 mol%, or from about 20 mol% to about 40 mol% of the total lipids present in the nucleic acid compositions (e.g., DNA or RNA compositions, particularly mRNA compositions) described herein.

In some embodiments of the nucleic acid (e.g., DNA or RNA) compositions (particularly mRNA compositions) described herein, when at least a portion of (i) the nucleic acid (e.g., DNA or RNA, particularly mRNA), (ii) the cationic/cationically ionizable lipid, and (iii) one or more additional lipids (e.g., one or more phospholipids and/or steroids) form a particle (e.g., LNP), the additional lipids (e.g., one or more phospholipids and/or steroids) can comprise from about 0 mol% to about 70 mol%, e.g., from about 2 mol% to about 60 mol%, from about 5 mol% to about 55 mol%, from about 5 mol% to about 50 mol%, from about 5 mol% to about 40 mol%, or from about 20 mol% to about 40 mol%, of the total lipids present in the particle.

Polymer-conjugated lipids Polymer-conjugated lipids are molecules that typically include a lipid portion and a polymer portion conjugated thereto.

An example of a polymer-conjugated lipid is a PEG-conjugated lipid, also referred to herein as a PEGylated lipid or PEG-lipid. The term "PEGylated lipid" refers to a molecule containing both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art.

Another example of a polymer-conjugated lipid is a polysarcosine-conjugated lipid, also referred to herein as a sarcosylated lipid or pSar-lipid. The term "sarcosylated lipid" refers to a molecule containing both a lipid portion and a polysarcosine (poly(N-methylglycine)) portion.

Other examples of polymer-conjugated lipids are polyoxazoline (POX)-conjugated and/or polyoxazine (POZ)-conjugated lipids, also referred to herein as conjugates of POX and/or POZ polymers with one or more hydrophobic chains, or oxazolinylated and/or oxazinylated lipids, or POX- and/or POZ-lipids. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule containing both a lipid portion and a polyoxazinyl portion. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule containing both a lipid portion and a polyoxazine portion. The term "oxazolinylated/oxazinylated lipid" or "POX/POZ-lipid" or "POXZ-lipid" refers to a molecule containing both a lipid portion and a polyoxazoline and polyoxazine copolymer portion.

As used herein, "polymer" refers to its conventional meaning, i.e., a molecular structure comprising one or more covalently bonded repeating units (monomers). The repeating units may all be identical, or in some cases, more than one type of repeating unit may be present in a polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties as described herein, may also be present in the polymer. If more than one type of repeating unit is present in a polymer, the polymer is referred to as a "copolymer." The repeating units forming the copolymer may be arranged in any manner. For example, the repeating units may be in random order, alternating order, or a "block" copolymer, i.e., an arrangement comprising one or more regions comprising a first repeating unit (e.g., a first block) and one or more regions (e.g., a second block) each comprising a second repeating unit, and so on. A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more different blocks.

In some embodiments, the polymer-conjugated lipid is designed to sterically stabilize the lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, the polymer-conjugated lipid can reduce the binding of such lipid particles to serum proteins and/or the resulting uptake by the reticuloendothelial system when administered in vivo.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein are substantially free of sarcosylated lipids.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein are substantially free of pegylated lipids having at least 30 consecutive ethylene glycol repeat units.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein are substantially free of (POX) conjugate and/or polyoxazine (POZ) conjugate lipids.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein are substantially free of pegylated lipids having at least 30 consecutive ethylene glycol repeat units, substantially free of sarcosylated lipids, and substantially free of (POX)-conjugated and/or polyoxazine (POZ)-conjugated lipids (or are free of pegylated lipids having at least 30 consecutive ethylene glycol repeat units, sarcosylated lipids, or oxazolinylated and/or oxazinylated lipids).

In some embodiments, the nucleic acid (e.g., RNA) compositions described herein are substantially free of any polymer-conjugated lipids other than the amphipathic OEG-conjugated compound (or free of any polymer conjugates other than the amphipathic OEG-conjugated compound).

PEG-Conjugated Lipids. In some embodiments, the nucleic acid compositions (e.g., DNA or RNA compositions, particularly mRNA compositions) described herein comprise a cationic/cationically ionizable lipid and a PEG-conjugated lipid. In some embodiments, the nucleic acid compositions (e.g., DNA or RNA compositions, particularly mRNA compositions) described herein can further comprise a neutral lipid (e.g., a phospholipid, cholesterol, or a derivative thereof) or a combination of neutral lipids (e.g., a phospholipid and cholesterol, or a derivative thereof). In some embodiments, the nucleic acid compositions (e.g., DNA or RNA compositions, particularly mRNA compositions) described herein comprise a cationic/cationically ionizable lipid described herein, a PEG-conjugated lipid, a neutral lipid (e.g., a phospholipid), and cholesterol, or a derivative thereof. In some embodiments, the phospholipid is DSPC. In some embodiments, the cationic/cationically ionizable lipid is a cationically ionizable lipid of Formula (X) (e.g., a cationically ionizable lipid of Formula (X-3) or (X-45)). In some embodiments, the cationic/cationically ionizable lipid is a cationically ionizable lipid of formula (XI) (e.g., a cationically ionizable lipid of formula (XIV-1), (XIV-2), or (XIV-3)). In some embodiments, the cationic/cationically ionizable lipid is DPL14, EA-2, or 3D-P-DMA.

In some embodiments, the PEG-conjugated lipid has the following general formula (XVI):
[In the ceremony:
Each of R ₁₂ and R ₁₃ is independently a straight or branched alkyl or alkenyl chain containing from 10 to 30 carbon atoms, where the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has an average value in the range of 30 to 60.
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof.

In certain embodiments of formula (XVI), R ₁₂ and R ₁₃ are each independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

In some embodiments of Formula (XVI), R ₁₂ and R ₁₃ are the same. In some embodiments, R ₁₂ and R ₁₃ are each a linear alkyl chain containing 12 carbon atoms. In some embodiments, R ₁₂ and R ₁₃ are each a linear alkyl chain containing 14 carbon atoms. In some embodiments, R ₁₂ and R ₁₃ are each a linear alkyl chain containing 16 carbon atoms.

In some embodiments of Formula (XVI), R ₁₂ and R ₁₃ are different. In some embodiments, one of R ₁₂ and R ₁₃ is a straight alkyl chain containing 12 carbon atoms, and the other of R ₁₂ and R ₁₃ is a straight alkyl chain containing 14 carbon atoms.

In some embodiments of Formula (XVI), w has an average value in the range of 40 to 50, for example, an average value of 45.

In some embodiments of Formula (XVI), w is within a range such that the PEG portion of the PEGylated lipid of Formula (XVI) has an average molecular weight of about 400 to about 6000 g/mol, e.g., about 1000 to about 5000 g/mol, about 1500 to about 4000 g/mol, or about 2000 to about 3000 g/mol.

Various PEG-conjugated lipids are known in the art, including PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω Examples of PEG-S-DMG include ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl) butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecaneoxy)propyl)carbamate or 2,3-di(tetradecaneoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate, but are not limited to these.

In some embodiments, the PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the PEGylated lipid has the following structure:

In some embodiments, the PEG-conjugated lipid is DMG-PEG 2000, for example, having the following structure:

In some embodiments, the PEG-conjugated lipid has the following structure:
where n ranges from 30 to 60, e.g., has an average value of about 50. In some embodiments, the PEG-conjugated lipid (PEGylated lipid) is _PEG2000 -C-DMA, which preferably refers to 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).

In some embodiments, the PEG-conjugated lipid is or includes one or more of the PEG-conjugated lipids or PEGylated lipids described in WO2017/075531 and WO2018/081480, the entire contents of which are incorporated herein by reference.

Sarcosylated Lipids In some embodiments, the sarcosylated lipid comprises 2 to 200 sarcosine units, e.g., 5 to 100 sarcosine units, 10 to 50 sarcosine units, 15 to 40 sarcosine units, e.g., about 23 sarcosine units.

In some embodiments, the sarcosylated lipid has the following general formula (XVII):
(wherein s is the number of sarcosine units).
Includes the structure of

In some embodiments, the sarcosylated lipid has the following general formula (XVIII):
wherein one of _R21 and _R22 comprises a hydrophobic group, the other is H, a hydrophilic group, or a functional group optionally comprising a targeting moiety; and x is the number of sarcosine units.
Includes the structure of

In some embodiments of Formula (XVIII), R ₂₁ is H, a hydrophilic group, or a functional group optionally containing a targeting moiety; and R ₂₂ comprises one or two linear alkyl or alkenyl groups, each having at least 12 carbon atoms, e.g., at least 14 carbon atoms. In some embodiments, each of the linear alkyl and alkenyl groups contains up to 30 carbon atoms, e.g., up to 28, up to 26, up to 24, up to 22, up to 20, or up to 18 carbon atoms. In some embodiments, R ₂₂ comprises one or two linear alkyl or alkenyl groups, each having 12 to 30 carbon atoms (e.g., 12 to 28 carbon atoms, 12 to 26 carbon atoms, 12 to 24 carbon atoms, 12 to 22 carbon atoms, 12 to 20 carbon atoms, or 12 to 18 carbon atoms).

In some embodiments, the sarcosylated lipid has the following general formula (IXX):
where R is H, a hydrophilic group, or a functional group optionally containing a targeting moiety; and s is the number of sarcosine units.
It has the following structure.

In some embodiments, the sarcosylated lipid has the following formula (IXX-1):
(Wherein, s1 is 23.)
The sarcosylated lipid of formula (IXX-1) is also referred to herein as "C14pSar23".

Oxazolinylated and/or oxazinylated lipids Oxazolinylated and/or oxazinylated lipids are conjugates comprising (i) a POX and/or POZ polymer and (ii) one or more hydrophobic chains.

In some embodiments, the POX and/or POZ polymer in the oxazolinylated and/or oxazinylated lipid has the following general formula (XX):
wherein a is an integer from 1 to 2; R ₁₁ is alkyl, particularly C _1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, independently selected at each repeat unit; and m refers to the number of POX and/or POZ repeat units.
Includes:

In some embodiments of the oxazolinylated and/or oxazinylated lipid, the POX and/or POZ polymer is a polymer of POX and has the following general formula (XXa):
It contains repeating units of

In some embodiments of the oxazolinylated and/or oxazinylated lipid, the POX and/or POZ polymer is a polymer of POZ and has the following general formula (XXb):
It contains repeating units of

In any of the above embodiments of formulas (XX), (XXa), and (XXb), m (i.e., the number of repeating units of formula (XXa) or formula (XXb) in the polymer) is preferably from 2 to 200.

In some embodiments of the oxazolinylated and/or oxazinylated lipids, the POX and/or POZ polymers have the following general formulas (XXa) and (XXb):
wherein the number of repeating units of formula (XXa) in the copolymer is 1 to 199; the number of repeating units of formula (XXb) in the copolymer is 1 to 199; and the sum of the number of repeating units of formula (XXa) and the number of repeating units of formula (XXb) in the copolymer is 2 to 200.
It is a copolymer containing repeating units of

In certain embodiments, the oxazolinylated and/or oxazinylated lipid has the following general formula (XXI) or (XXI'):
[In the ceremony:
a is an integer from 1 to 2;
R ₁₁ is alkyl, particularly C _1-3 alkyl, for example methyl, ethyl, iso-propyl or n-propyl, independently selected for each repeat unit;
m is 2 to 200;
R ₁₂ is R ₁₄ or -L ₁₁ (R ₁₄ ) _p , where each R ₁₄ is independently a hydrocarbyl group; L ₁₁ is a linker; and p is 1 or 2; and R ₁₃ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, where the C _1-6 alkyl group is optionally -OH, SH, halogen, -CN, -N ₃ , C R ₂₀ is selected from the group consisting of H, C _1-3 alkyl, and _{3- to} 6-membered heterocyclyl, wherein _{each of the C 1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N 3 , C 2-6 alkynyl, -COOH, -NR 22 R 23 , sugars , amino acids , peptides ,} and members of a targeting pair; R ₂₁ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein _C Each of _{the 1-6} alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ₂₂ R ₂₃ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ₂₂ and R ₂₃ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with -OH, SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C and is substituted with one or more substituents independently selected from the group consisting of —N(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair.
In formula (XXI), R ₁₂ is bonded to the N-terminus (i.e., the terminal N atom) of the POX and/or POZ polymer and R ₁₃ is bonded to the C-terminus (i.e., the terminal C atom) of the POX and/or POZ polymer, and in one mode (XXI'), R ₁₂ is bonded to the C-terminus (i.e., the terminal C atom) of the POX and/or POZ polymer and R ₁₃ is bonded to the N-terminus (i.e., the terminal N atom) of the POX and/or POZ polymer.

In some embodiments, the oxazolinylated and/or oxazinylated lipid has the following general formula (XXIII):
R ₁₅ -POXZ-R ₁₆
[In the ceremony:
R ₁₅ is R ₁₇ or -L ₁₂ (R ₁₇ ) _q , where each R ₁₇ is independently a hydrocarbyl group; L ₁₂ is a linker; and q is 1 or 2;
POXZ has the following general formulas (XXa) and (XXb):
wherein each R ₁₁ is independently alkyl, particularly C _1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected in each repeat unit; the number of repeat units of formula (XXa) in the copolymer is 1 to 199; the number of repeat units of formula (XXb) in the copolymer is 1 to 199; the sum of the number of repeat units of formula (XXa) and the number of repeat units of formula (XXb) in the copolymer is 2 to 200; and the repeat units of formulas (XXa) and (XXb) are arranged randomly, periodically, alternatingly, or in a block manner; and R ₁₆ is H, C _1-6 alkyl, C _2-6 alkynyl, —OR ²⁰ , —SR ²⁰ , halogen, —CN, —N ₃ , —OC(O)R ²¹ , —C(O)R ²¹ , —NR ²² R ²³ , —COOH, —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —NR ₂₂ R ₂₃ , —C(O)NR ₂₂ R ₂₃ , —NR ₂₂ C(O)R ₂₁ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ₂₀ is selected from the group consisting of H, C _1-3 alkyl, and 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of —OH, SH, halogen, —CN, —N ₃ , C R ₂₁ is substituted with one or more substituents independently selected from the group consisting of _{C 1-6} alkyl and _3-6 membered heterocyclyl, wherein _each of the C _1-6 alkyl and 3-6 membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from _the group consisting of -OH, SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ₂₂ R ₂₃ , sugar, amino acid, peptide, and member of a targeting pair; and R ²² and R ²³ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ₂₂ and R ₂₃ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, SH, halogen, -CN, _-N , C _{2-6 alkynyl} , -COOH, _-NH , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) , a sugar, an amino acid, a peptide, and a member of a targeting pair.
It has.

Conjugates of (a) an amphiphilic OEG conjugate compound and (b) a compound comprising another member of a targeting pair The amphiphilic OEG conjugate compounds of the present invention comprising a member of a targeting pair can be used to prepare conjugates comprising (a) the amphiphilic OEG conjugate compound and (b) a compound comprising another member of a targeting pair.

Thus, the present invention provides (a) an amphiphilic OEG conjugate compound of the present disclosure (particularly a compound of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IX b), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), ( V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V- 13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21) , (V-22), (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V -30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38 ), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), ( V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-5 The present invention also relates to conjugates of a compound having one of the formulas (IXa), (IXi), (IXm), (V-1), (V-5), (V-17), and (V-25), preferably having any of the formulas (IXa), (IXi), and (IXm) or any of the formulas (V-1), (V-5), (V-17), and (V-25), and (b) another member of the targeting pair.

In certain embodiments of amphiphilic OEG conjugate compounds comprising members of a targeting pair (particularly those of formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Ve'), j), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (I Xc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp ), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-1 1), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22) , (V-23), (V-24), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), ( V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-4 5), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably having any of the formulas (IXa), (IXi), ( (IXm), (V-1), (V-5), (V-17) and (V-25), for example, any of formulas (IXa), (IXi) and (IXm) or any of formulas (V-1), (V-5), (V-17) and (V-25), the targeting pair is selected from the group consisting of the following pairs: maleimide-thiol; thiol-halogenated (particularly halogenated) conjugated diene-substituted alkenes (dienophiles) (especially in Diels-Alder reactions); antigen - an antibody specific for the antigen (including an immunoreactive fragment or derivative thereof such as a Fab, Fab', F(ab')2 or scFv fragment or a single domain antibody, e.g., a VHH fragment or a nanobody); biotin - streptavidin; biotin - avidin; biotin - neutravidin; folate - folate receptor; transferrin - transferrin receptor; aptamer - a molecule for which the aptamer is specific (e.g., pegaptanib - VEGF receptor); arginine-glycine-aspartic acid (RGD) peptide - α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide - aminopeptidase N; galactose - asialoglycoprotein receptor.

For example, if an amphiphilic OEG conjugate compound contains a maleimide moiety as a member of a targeting pair (e.g., as a component of ^R3 ), then the compound containing the other member of the targeting pair contains a thiol moiety. Reaction of the maleimide moiety with the thiol moiety results in covalent attachment of the amphiphilic OEG conjugate compound to the compound containing the other member of the targeting pair via the succinimidyl-S-moiety, i.e., providing a conjugate between the amphiphilic OEG conjugate compound and the compound containing the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphiphilic OEG conjugate compound of formula (V) and a thiol compound ("HS-compound") is shown below.
[In the formula, R ² , X ¹ , X ² , Y, z and n (especially the formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vg), ( Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (Vf'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIII With respect to any of formulas (IXa), (IXi), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq) and (IXr), preferably with respect to any of formulas (IXa), (IXi) and (IXm), as defined above.
In some embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In another example, the amphiphilic OEG conjugate compound comprises a thiol moiety as a member of a targeting pair (e.g., as a component of ^R3 ), and the compound comprising the other member of the targeting pair comprises a halogenated (particularly brominated) alkyl group. Reaction of the thiol moiety with the halogenated (particularly brominated) alkyl group results in covalent bonding of the amphiphilic OEG conjugate compound to the compound comprising the other member of the targeting pair via the thioether moiety, i.e., providing a conjugate between the amphiphilic OEG conjugate compound and the compound comprising the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphiphilic OEG conjugate compound of formula (V) and a brominated alkane ("Br-alkyl compound") is shown below.
[In the formula, R ² , X ¹ , X ² , Y, z and n (especially the formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIII With respect to any of formulas (IXa), (IXi), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq) and (IXr), preferably with respect to any of formulas (IXa), (IXi) and (IXm), as defined above.
In certain embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In certain embodiments, n is 16. In certain embodiments, n is 14. In certain embodiments, n is 12. In certain embodiments, n is 10. In certain embodiments, n is 8. In certain embodiments, z is 2 to 7. In certain embodiments, z is 2 to 5. In certain embodiments, z is 2 or 3. In certain embodiments, z is 2. Exemplary reaction schemes for reactions using reverse targeting members (i.e., amphiphilic OEG conjugate compounds containing halogenated (particularly brominated) alkyl groups; and thiol compounds ("HS-compounds")) are shown below.

In another example, the amphipathic OEG conjugate compound comprises an antigen (e.g., a peptide antigen) as a member of a targeting pair (e.g., as a component of ^R3 ), and the compound comprising the other member of the targeting pair comprises an antibody specific to the antigen (including an immunoreactive fragment or derivative thereof, such as a Fab, Fab', F(ab')2, or scFv fragment, or a single domain antibody, e.g., a VHH fragment, or a nanobody). The reaction between the antigen and the antibody results in non-covalent binding of the amphipathic OEG conjugate compound to the compound comprising the other member of the targeting pair via the thioether moiety, i.e., providing a conjugate between the amphipathic OEG conjugate compound and the compound comprising the other member of the targeting pair. An exemplary reaction scheme for this reaction using an amphipathic OEG conjugate compound of formula (V) comprising an antigen and an antibody specific to the antigen is shown below.
[In the formula, R ² , X ¹ , X ² , Y, z and n (especially the formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (V h), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (I (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq) and (IXr), preferably with respect to any of formulas (IXa), (IXi) and (IXm), as defined above; Pept is an antigen; Antibody is an antibody specific for the antigen; and the dashed line represents a non-covalent bond between the antigen and the antibody.
In some embodiments, n is 5 to 25, preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments, the conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has the following formulae (CI), (CII), and (CIII):
[In the formula, R ² , X ¹ , X ² , Y, z and n (especially the formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), ( Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), ( Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (I is as defined above for any of formulas (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), and (IXr), preferably for any of formulas (IXa), (IXi), and (IXm); each m1 is independently 1, 2, 3, 4, or 5 (e.g., 1, 2, 3, 4, preferably 1, 2, or 3, more preferably 1 or 2); compound is any compound (e.g., an antigen or an antibody specific for the antigen); Pept is an antigen; and antibody is any compound including an antibody specific for the antigen.
In some embodiments, the antigen is a peptide, for example, a peptide having one of the following sequences: PSRLEEELRRRLTE-NH ₂ (SEQ ID NO: 11), SRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 12), or PSRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 13) (in SEQ ID NOs: 12 and 13, "cyclo" means that the side chains of the two amino acids at positions 1 and 5 within the brackets (i.e., E and K) join together to form an amide bond). In some embodiments, n is 5 to 25, preferably 8, 10, or 14. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments, the conjugates of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair are represented by the following formulas (Ca), (Cb), and (Cc):
[In the formula, R ² , X ¹ , X ² , Y, z and n (especially the formulas (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg' ), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), ( (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq) and (IXr), preferably as defined above for any of formulas (IXa), (IXi) and (IXm); and the compound is any compound (e.g., an antigen or an antibody specific for said antigen).
In some embodiments, the compound is an antigen, preferably a peptide, for example, a peptide having one of the following sequences: PSRLEEELRRRLTE-NH ₂ (SEQ ID NO: 11), SRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 12), or PSRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 13) (in SEQ ID NOs: 12 and 13, "cyclo" means that the side chains of the two amino acids at positions 1 and 5 within the brackets (i.e., E and K) join together to form an amide bond). In some embodiments, n is 5 to 25, and preferably 8, 10, 12, 14, or 16. In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

In some embodiments, the conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair has the following formulae (CIV) and (CV):
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); each of m1 and m2 is independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. In certain embodiments of Formula (CIV), m1 is 2, 3, or 4 (preferably 2); and m2 is 2, 3, or 4 (preferably 2). In certain embodiments of Formula (CV), m1 is 1 and m2 is 2; or m1 is 2 and m2 is 1. In certain embodiments, the antigen is a peptide, for example, a peptide having one of the following sequences: PSRLEEELRRRLTE-NH ₂ (SEQ ID NO:11), SRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO:12), PSRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO:13). In certain embodiments, n is 16. In certain embodiments, n is 14. In certain embodiments, n is 12. In certain embodiments, n is 10. In certain embodiments, n is 8.

In some embodiments, the conjugates of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair have the following formulae (Ce), (Cf), and (Cg):
wherein n is 5 to 25, preferably 8, 10, 12, ₁₄ , or 16; in each case, --C(O) _{C.sub.17H.sub.35} refers to the moiety --C(O)( _CH.sub.2 ) _{.sub.16CH.sub.3} ( _stearoyl ); and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. In some embodiments, the antigen is a peptide, for example, a peptide having one of the following sequences: PSRLEEELRRRLTE-NH ₂ (SEQ ID NO: 11), SRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 12), PSRLE-cyclo(EELRK)-RLTE-NH ₂ (SEQ ID NO: 13). In some embodiments, n is 16. In some embodiments, n is 14. In some embodiments, n is 12. In some embodiments, n is 10. In some embodiments, n is 8.

A specific example of a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair is the following compound (C1):
where n is 14; and Pept is a peptide having the sequence PSRLEEELRRRLTE- _NH2 .
or a salt thereof.

In a further aspect, the present invention provides a nucleic acid conjugate composition comprising: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair (e.g., a conjugate of any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)) disclosed herein.

It is understood that any embodiment described in the context of a nucleic acid composition comprising an amphipathic OEG conjugate compound (including the use of a nucleic acid composition comprising an amphipathic OEG conjugate compound) also applies to any embodiment of a nucleic acid conjugate composition.

Thus, in some embodiments, the nucleic acid conjugate composition comprises (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid; and (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair (e.g., a conjugate of any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)) disclosed herein.

In some embodiments, the nucleic acid conjugate composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid selected from the group consisting of DODMA, DOTMA, DPL14, 3D-P-DMA, Formula (X), and Formula (XI); (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair and (b) a compound comprising the other member of the targeting pair disclosed herein (e.g., a conjugate of any of Formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)); and (iv) one or more additional lipids. In some embodiments, the one or more additional lipids are selected from neutral lipids and combinations thereof. In some embodiments, the neutral lipids include phospholipids, steroid lipids, and combinations thereof. In some embodiments, the one or more additional lipids are a combination lipid of a phospholipid and a steroid.

In some embodiments, the nucleic acid conjugate composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any of Formulas A-G; (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair disclosed herein and (b) a compound comprising the other member of the targeting pair (e.g., a conjugate of any of Formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)); (iv) a phospholipid; and (v) cholesterol.

In some embodiments, the nucleic acid conjugate composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (X); (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair disclosed herein and (b) a compound comprising the other member of the targeting pair (e.g., a conjugate of any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)); (iv) a phospholipid; and (v) cholesterol.

In some embodiments, the nucleic acid conjugate composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of formula (XI) (e.g., any of formulas (XIIa), (XIIb), (XIIIa), (XIIIb), (XIV-1), (XIV-2), and (XIV-3)); (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair disclosed herein and (b) a compound comprising the other member of the targeting pair (e.g., any of conjugates of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)).

In some embodiments, the nucleic acid conjugate composition comprises: (i) a nucleic acid (e.g., DNA or RNA); (ii) a cationic or cationically ionizable lipid of any of formulas (XV-1) to (XV-6); (iii) a conjugate of (a) an amphiphilic OEG conjugate compound comprising a member of a targeting pair disclosed herein and (b) a compound comprising the other member of the targeting pair (e.g., a conjugate of any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1)); (iv) a phospholipid; and (v) cholesterol.

In some embodiments of the nucleic acid conjugate composition, the nucleic acid is DNA.

In some embodiments of the nucleic acid conjugate composition, the nucleic acid is RNA, particularly mRNA.

In some embodiments of the nucleic acid conjugate composition, the composition is a pharmaceutical composition.

In some embodiments of the nucleic acid conjugate composition, the composition, particularly the pharmaceutical composition, is a vaccine.

In some embodiments of the nucleic acid conjugate composition, the composition, particularly a pharmaceutical composition, further comprises one or more pharmaceutically acceptable carriers, diluents, and/or additives.

In some embodiments of the nucleic acid conjugate composition, the nucleic acid (e.g., DNA or RNA) and/or composition, particularly a pharmaceutical composition, is a component of a kit.

In some embodiments of the nucleic acid conjugate composition, the kit further includes instructions for using the nucleic acid (e.g., DNA or RNA) to induce an immune response in a subject against a pathogen, e.g., a virus such as a coronavirus. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments of the nucleic acid conjugate composition, the kit further includes instructions for use of the nucleic acid (e.g., DNA or RNA) for therapeutic or prophylactic treatment of an infection, e.g., a viral infection, such as a coronavirus infection, in a subject. In some embodiments, the coronavirus is a betacoronavirus. In some embodiments, the coronavirus is a sarbecovirus. In some embodiments, the coronavirus is SARS-CoV-2.

In some embodiments of the nucleic acid conjugate composition, the subject is a human.

In some embodiments of nucleic acid conjugate compositions, the nucleic acid (e.g., DNA or RNA) in a nucleic acid (e.g., DNA or RNA) composition described herein (e.g., in a nucleic acid (e.g., DNA or RNA) particle) is at a concentration of about 0.002 mg/mL to about 5 mg/mL, about 0.002 mg/mL to about 2 mg/mL, about 0.005 mg/mL to about 2 mg/mL, about 0.01 mg/mL to about 1 mg/mL, about 0.05 mg/mL to about 0.5 mg/mL, or about 0.1 mg/mL to about 0.5 mg/mL. In specific embodiments, the nucleic acid (e.g., DNA or RNA) is at a concentration of about 0.005 mg/mL to about 0.1 mg/mL, about 0.005 mg/mL to about 0.09 mg/mL, about 0.005 mg/mL to about 0.08 mg/mL, about 0.005 mg/mL to about 0.07 mg/mL, about 0.005 mg/mL to about 0.06 mg/mL, or about 0.005 mg/mL to about 0.05 mg/mL.

In a further aspect, the present invention provides a method for delivering a nucleic acid to a cell in a subject, the method comprising administering to the subject a nucleic acid conjugate composition as specified herein.

In a further aspect, the present invention provides a method for delivering a therapeutic peptide or protein to a subject, comprising administering to the subject a nucleic acid conjugate composition as defined herein, wherein the nucleic acid encodes the therapeutic peptide or protein.

In a further aspect, the present invention provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid conjugate composition specified herein, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the present invention provides a nucleic acid conjugate composition specified herein for use in a method of treating or preventing a disease or disorder in a subject, wherein delivery of the nucleic acid to cells of the subject is beneficial to treating or preventing the disease or disorder.

In a further aspect, the present invention provides a method of treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a nucleic acid conjugate composition specifically disclosed herein, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder. In a related aspect, the present invention provides a nucleic acid conjugate composition specifically disclosed herein for use in a method of treating or preventing a disease or disorder in a subject, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial to treating or preventing the disease or disorder.

[0023] In certain embodiments of nucleic acid (e.g., DNA or RNA) composition embodiments, the one or more additional lipids comprise one of the following components: (1) a neutral lipid; (2) a steroid; or (3) a mixture of a neutral lipid and a steroid, each at the concentrations described above. In certain embodiments, the one or more additional lipids comprise (3) a mixture of a neutral lipid and a steroid (and preferably does not comprise a PEG-lipid having at least 30 consecutive ethylene glycol repeat units, more preferably does not comprise any polymer-conjugated lipid other than an amphiphilic OEG-conjugated compound). In certain embodiments, the one or more additional lipids comprise one of the following components: (1) a phospholipid; (2) cholesterol; or (3) a mixture of a phospholipid and cholesterol, each at the concentrations described above. In certain embodiments, the one or more additional lipids comprise (3) a mixture of a phospholipid and cholesterol (and preferably does not comprise a PEG-lipid having at least 30 consecutive ethylene glycol repeat units, more preferably does not comprise any polymer-conjugated lipid).

Thus, in a preferred embodiment, the nucleic acid compositions described herein comprise (i) a nucleic acid (e.g., DNA or RNA, particularly mRNA); (ii) a cationic or cationically ionizable lipid; (iiia) an amphipathic OEG conjugate compound or (iiib) a conjugate of an amphipathic OEG conjugate compound comprising a member of a targeting pair disclosed herein and a compound comprising the other member of the targeting pair; (iv) a steroid; and (v) a neutral lipid (particularly a phospholipid), preferably each at the concentrations described above.

In a specific embodiment, the amphiphilic OEG conjugate compound has the formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI), (VI'), (VII), (VII'), (VIII), (VIII') , (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), (IXj), (IXk), (IXl), (IXm ), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V -11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24) ), (V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V -38), (V-39), (V-40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51) , (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63).

In particularly preferred embodiments, the amphiphilic OEG conjugate compound has any of formulas (V-1), (V-5), (V-17), and (V-25).

The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

In some embodiments, the nucleic acid (e.g., DNA or RNA) is present in a nucleic acid composition (e.g., a DNA or RNA composition) described herein at a concentration of about 1 mg/L to about 500 mg/L, e.g., about 1 mg/L to about 100 mg/L, about 5 mg/L to about 100 mg/L, or about 10 mg/L to about 100 mg/L.

In one embodiment, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the amphiphilic OEG conjugate compound is represented by the formula (V), (V'), (Va), (Va'), (Vb), (Vb'), (Vc), (Vc'), (Vd), (Vd'), (Ve), (Ve'), (Vf), (Vf'), (Vg), (Vg'), (Vh), (Vh'), (Vi), (Vi'), (Vj), (Vj'), (VI) , (VI'), (VII), (VII'), (VIII), (VIII'), (VIIIa), (VIIIa'), (IXa), (IXb), (IXc), (IXd), (IXe), (IXf), (IXg), (IXh), (IXi), ( IXj), (IXk), (IXl), (IXm), (IXn), (IXo), (IXp), (IXq), (IXr), (V-1), (V-2), (V-3), (V-4), (V-5), (V-6), (V-7), (V-8), (V-9), (V-10), (V-11), (V-12), (V-13), (V-14), (V-15), (V-16), (V-17), (V-18), (V-19), (V-20), (V-21), (V-22), (V-23), (V-24), ( V-25), (V-26), (V-27), (V-28), (V-29), (V-30), (V-31), (V-32), (V-33), (V-34), (V-35), (V-36), (V-37), (V-38), (V-39), (V- 40), (V-41), (V-42), (V-43), (V-44), (V-45), (V-46), (V-47), (V-48), (V-49), (V-50), (V-51), (V-52), (V-53), (V-54), (V-55), (V-56), (V-57), (V-58), (V-59), (V-60), (V-61), (V-62) and (V-63), preferably any of formulas (V-1), (V-5), (V-17) and (V-25).

In specific embodiments of the nucleic acid conjugate composition, the conjugate has any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1).

In some embodiments of the nucleic acid conjugate composition, the steroid is cholesterol; the neutral lipid is selected from the group consisting of DSPC, DPPC, DSPE, and DPPE; and the conjugate has any of formulas (CI), (CII), (CIII), (CIV), (CV), (Ca), (Cb), (Cc), (Ce), (Cf), (Cg), and (C1).

In nucleic acid conjugate compositions, the N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

In some embodiments of nucleic acid conjugate compositions, the nucleic acid (e.g., DNA or RNA) is present in a nucleic acid composition (e.g., a DNA or RNA composition) described herein at a concentration of about 1 mg/L to about 500 mg/L, e.g., about 1 mg/L to about 100 mg/L, about 5 mg/L to about 100 mg/L, or about 10 mg/L to about 100 mg/L.

Compositions Comprising Nucleic Acid (e.g., DNA or RNA, preferably mRNA) Particles The nucleic acid (e.g., DNA or RNA) compositions described herein (including the nucleic acid conjugate compositions described herein) can comprise nucleic acid particles (e.g., DNA or RNA particles, such as RNA LNPs), preferably a plurality of nucleic acid particles (e.g., a plurality of DNA or RNA particles). The term "plurality of nucleic acid particles" or "plurality of nucleic acid-lipid particles" refers to a population of a number of particles. In certain embodiments, the term refers to a population of 10, 10, 10, ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , 10, ¹⁰ , ¹⁰ , ¹⁰ , 10, ¹⁰ , ¹⁰ , ¹⁰ , 10, ¹⁰ , ¹⁰ , 10, ¹⁰ , 10, ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , ¹⁰ , or ¹⁰ particles or more.

The nucleic acid particles (e.g., DNA or RNA particles, such as RNA-containing LNPs (or "RNA LNPs")) described herein, in some embodiments, have a diameter of about 30 nm to about 1000 nm, about 30 nm to about 800 nm, about 30 nm to about 700 nm, about 30 nm to about 600 nm, about 30 nm to about 500 nm, about 30 nm to about 450 nm, about 30 nm to about 400 nm, about 30 nm to about 350 nm, about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 190 nm, about 30 nm to about 18 ...250 nm, about 30 nm to about 200 nm, about 30 nm to about 300 nm, about 30 nm to about 400 nm, about 30 nm to about 450 nm, about 30 nm to about 400 nm, about 30 nm to about 450 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 190 nm, about 30 nm to about 180 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 250 nm, about 30 nm to about 20 The average diameter ranges from about 50 nm to about 170 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 190 nm, about 50 nm to about 180 nm, about 50 nm to about 170 nm, about 50 nm to about 160 nm, or about 50 nm to about 150 nm.

In some embodiments, the nucleic acid particles (e.g., DNA or RNA particles, such as RNA LNPs) described herein have an average diameter ranging from about 40 nm to about 800 nm, about 50 nm to about 700 nm, about 60 nm to about 600 nm, about 70 nm to about 500 nm, about 80 nm to about 400 nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, or about 200 nm to about 400 nm.

In some embodiments, the nucleic acid particles (e.g., DNA or RNA particles, such as RNA LNPs) described herein can have an average diameter ranging from about 30 nm to about 500 nm, e.g., from about 30 nm to about 200 nm or from about 50 nm to about 150 nm.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein comprise particles having a size of at least 10 μm in an amount of 4000/ml, preferably at most 3500/ml, e.g., at most 3400/ml, at most 3300/ml, at most 3200/ml, at most 3100/ml, or at most 3000/ml.

It will be apparent to one skilled in the art that the plurality of particles may comprise any fraction of the above ranges or any range therein.

For example, the nucleic acid particles described herein (e.g., DNA or RNA particles, such as RNA LNPs) produced by the methods described herein exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or about 0.05 or less. Illustratively, the nucleic acid particles (e.g., DNA or RNA LNPs) exhibit a polydispersity index ranging from about 0.05 to about 0.2, e.g., from about 0.05 to about 0.1.

Generally, the nucleic acid particles (e.g., DNA or RNA particles such as RNA LNPs) described herein are "nucleic acid-lipid particles" (e.g., "DNA-lipid particles" or "RNA-lipid particles") that can be used to deliver nucleic acids to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid-lipid particles are typically formed from a cationic or cationically ionizable lipid (e.g., a cationic or cationically ionizable lipid disclosed herein), an amphipathic OEG conjugate compound disclosed herein (or a conjugate of an amphipathic OEG conjugate compound comprising a member of a targeting pair and a compound comprising the other member of the targeting pair), and one or more additional lipids (e.g., a phospholipid disclosed herein (e.g., DSPC) and a steroid (e.g., cholesterol or an analog thereof)).

Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable lipid, the amphiphilic OEG conjugate compound (or its conjugate), and one or more additional lipids combine with nucleic acid (e.g., DNA or RNA) to form particles that are colloidally stable due to the presence of the amphiphilic OEG conjugate compound (or its conjugate), particularly the stealth properties of the amphiphilic OEG conjugate compound (or its conjugate).

In some embodiments, a nucleic acid-lipid particle (e.g., a DNA- or RNA-lipid particle) contains more than one type of nucleic acid molecule (e.g., more than one type of DNA or RNA molecule), where the molecular parameters of the nucleic acid molecules may be similar to one another or may differ, such as with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions, or other characteristics.

In some embodiments, the compositions described herein are liquid or solid, where solid refers to a dry, lyophilized, or frozen form.

The compositions described herein may also include cryoprotectants and/or surfactants as stabilizers to avoid substantial loss of product quality, particularly substantial loss of nucleic acid (e.g., DNA or RNA) activity, during storage and/or freezing, e.g., reducing or preventing aggregation, particle collapse, nucleic acid (e.g., DNA or RNA) degradation, and/or other types of damage.

In some embodiments, the cryoprotectant is a carbohydrate. As used herein, the term "carbohydrate" includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

In some embodiments, the cryoprotectant is a monosaccharide. As used herein, the term "monosaccharide" refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed into simpler carbohydrate units. Exemplary monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose, etc.

In some embodiments, the cryoprotectant is a disaccharide. As used herein, the term "disaccharide" refers to a compound or chemical moiety formed from two monosaccharide units linked together via a glycosidic bond, e.g., a 1-4 or 1-6 bond. A disaccharide can be hydrolyzed into two monosaccharides. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose, etc.

The term "trisaccharide" means three sugars linked together to form one molecule. Examples of trisaccharides include raffinose and melezitose.

In some embodiments, the cryoprotectant is an oligosaccharide. As used herein, the term "oligosaccharide" refers to a compound or chemical moiety formed by 3 to about 15, preferably 3 to about 10, monosaccharide units linked together via glycosidic bonds, e.g., 1-4 or 1-6 bonds, to form a linear, branched, or cyclic structure. Exemplary oligosaccharide cryoprotectants include cyclodextrin, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. Oligosaccharides can be oxidized or reduced.

In some embodiments, the cryoprotectant is a cyclic oligosaccharide. As used herein, the term "cyclic oligosaccharide" refers to a compound or chemical moiety formed by 3 to about 15, preferably 6, 7, 8, 9, or 10, monosaccharide units linked together via glycosidic bonds, e.g., 1-4 or 1-6 bonds, to form a ring structure. Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as α-cyclodextrin, β-cyclodextrin, or γ-cyclodextrin.

Other exemplary cyclic oligosaccharide cryoprotectants include compounds that include a cyclodextrin moiety in a larger molecular structure, such as a polymer, that includes the cyclic oligosaccharide moiety. The cyclic oligosaccharide can be oxidized or reduced, e.g., oxidized to the dicarbonyl form. As used herein, the term "cyclodextrin moiety" refers to a cyclodextrin (e.g., α-, β-, or γ-cyclodextrin) radical that is included in or is part of a larger molecular structure, such as a polymer. The cyclodextrin moiety can be attached to one or more other moieties directly or via an optional linker. The cyclodextrin moiety can be oxidized or reduced, e.g., oxidized to the dicarbonyl form.

A carbohydrate cryoprotectant, e.g., a cyclic oligosaccharide cryoprotectant, can be a derivatized carbohydrate. For example, in some embodiments, the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-β-cyclodextrin, e.g., a partially etherified cyclodextrin (e.g., a partially etherified β-cyclodextrin).

Exemplary cryoprotectants are polysaccharides. As used herein, the term "polysaccharide" refers to a compound or chemical moiety formed by at least 16 monosaccharide units linked via glycosidic bonds, e.g., 1-4 or 1-6 bonds, to form a linear, branched, or cyclic structure, and includes polymers that contain polysaccharides as part of their backbone structure. In the backbone, the polysaccharides can be linear or cyclic. Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin, and the like.

In some embodiments, the cryoprotectant is a sugar alcohol. As used herein, the term "sugar alcohol" refers to an organic compound containing at least two carbon atoms and one hydroxyl group attached to each carbon atom. Typically, sugar alcohols are derived from sugars (e.g., by hydrogenation of sugars) and are water-soluble solids. As used herein, the term "sugar" refers to a sweet, soluble carbohydrate. Examples of sugar alcohols include ethylene glycol, glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt, maltitol, lactitol, maltotriitol, maltotetriitol, and polyglycitol. In some embodiments, the sugar alcohol has the formula HOCH ₂ (CHOH) _n CH ₂ OH, where n is 0-22 (e.g., 0, 1, 2, 3, or 4), or is a cyclic variant thereof (which may be derived formally by dehydrating the sugar alcohol to provide a cyclic ether; for example, isosorbide is a cyclic dehydrated variant of sorbitol).

In some embodiments, the cryoprotectant is glycerol and/or sorbitol.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions (e.g., RNA LNP compositions) described herein may include sucrose as a cryoprotectant. Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the composition, thereby preventing aggregation of nucleic acid (particularly DNA or RNA) particles and maintaining the chemical and physical stability of the composition. In some embodiments, alternative cryoprotectants to sucrose are contemplated in the present invention. Alternative stabilizing agents include, but are not limited to, glucose, glycerol, and sorbitol.

Preferred cryoprotectants are selected from the group consisting of sucrose, glucose, glycerol, sorbitol, and combinations thereof. In a preferred embodiment, the cryoprotectant comprises sucrose and/or glycerol. In a more preferred embodiment, the cryoprotectant is sucrose.

In certain other embodiments, the nucleic acid (e.g., DNA or RNA) compositions (e.g., RNA LNP compositions) described herein are substantially free of cryoprotectants, e.g., free of any cryoprotectants.

Some embodiments of the present invention contemplate the use of a chelating agent in the nucleic acid (e.g., DNA or RNA) compositions (e.g., RNA LNP compositions) described herein. A chelating agent is a chemical compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby producing a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise accelerate RNA degradation in the present invention. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, calcium pentetate, sodium pentetate, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycol ether-N,N,N',N'-tetraacetic acid. In some embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate. In some embodiments, the EDTA is at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 2.5 mM, or about 0.25 mM to about 1 mM.

In some embodiments, the aqueous phase of a nucleic acid (e.g., DNA or RNA) composition (e.g., an RNA LNP composition) described herein does not include a chelating agent. For example, if a nucleic acid (e.g., DNA or RNA) composition (e.g., an RNA LNP composition) described herein includes a chelating agent, the chelating agent, if present, is preferably present only in the particles.

In some embodiments, the compositions described herein contain water as a major component and/or the total amount of solvents other than water in the composition is less than about 1.0% (v/v). For example, the amount of water in the composition can be at least 50% (w/w), e.g., at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), at least 90% (w/w), or at least 95% (w/w). In particular, when the composition includes a cryoprotectant, the amount of water contained in the composition can be at least 50% (w/w), e.g., at least 55% (w/w), at least 60% (w/w), at least 65% (w/w), at least 70% (w/w), at least 75% (w/w), at least 80% (w/w), at least 85% (w/w), or at least 90% (w/w). If the composition is substantially free of a cryoprotectant, the amount of water contained in the composition can be at least 95% (w/w). Additionally or alternatively, the total amount of non-aqueous solvents in the composition may be less than about 1.0% (v/v), e.g., less than about 0.9% (v/v), less than about 0.8% (v/v), less than about 0.7% (v/v), less than about 0.6% (v/v), less than about 0.5% (v/v), less than about 0.4% (v/v), less than about 0.3% (v/v), less than about 0.2% (v/v), less than about 0.1% (v/v), less than about 0.05% (v/v), or less than about 0.01% (v/v). In this regard, a cryoprotectant that is liquid under normal conditions is considered a cryoprotectant, not a non-aqueous solvent. In other words, the optional limitation above that the total amount of non-aqueous solvents in the composition may be less than about 1.0% (v/v) does not apply to a cryoprotectant that is liquid under normal conditions.

Pharmaceutical Compositions Compositions comprising nucleic acids (e.g., DNA or RNA), such as the nucleic acid (e.g., DNA or RNA) particles (particularly RNA LNPs) described herein, are useful as or for the preparation of pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment. Thus, in some embodiments, the compositions described herein, particularly the nucleic acid compositions described herein, are pharmaceutical compositions.

The nucleic acid (e.g., DNA or RNA) compositions described herein can be administered in the form of any suitable pharmaceutical composition.

The term "pharmaceutical composition" refers to a composition comprising a therapeutically active agent, preferably together with a pharmaceutically acceptable carrier, diluent, and/or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. In the present invention, the pharmaceutical composition comprises a nucleic acid (e.g., DNA or RNA), such as a nucleic acid (e.g., DNA or RNA) particle (particularly an RNA LNP), as described herein.

The pharmaceutical compositions of the present invention may contain or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Chemokines can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IFN-α, IFN-γ, GM-CSF, or LT-α. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present invention include lipopeptides, such as Pam3Cys, as well as lipophilic components such as saponin, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid A (MPL), monomycoloylglycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

The pharmaceutical compositions of the present invention may be in a storable form (e.g., frozen or dried or lyophilized/freeze-dried form) or in a "ready-to-use form" (i.e., a form, particularly a liquid form, that can be immediately administered to a subject without any processing, e.g., thawing, reconstitution, or dilution). Thus, prior to administration of a pharmaceutical composition in a storable form, the storable form must be processed or transferred to a ready-to-use or administrable form. For example, a frozen pharmaceutical composition must be thawed. Ready-to-use injections can be provided in containers such as vials, ampoules, or syringes, where the container may contain one or more doses.

The pharmaceutical compositions of the present invention are generally provided in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

The term "pharmaceutically acceptable" refers to a substance that is non-toxic and does not interact with the action of the active ingredient(s) of a pharmaceutical composition.

The term "pharmaceutically effective amount" refers to an amount that, alone or together with further doses, achieves the desired response or desired effect. In the case of treatment of a particular disease, the desired response preferably relates to inhibition of the course of the disease. This includes slowing the progression of the disease, and particularly halting or reversing the progression of the disease. The desired response in disease treatment can also be delaying or preventing the onset of the disease or condition. The effective amount of the particles or pharmaceutical compositions described herein will depend on the condition being treated, the severity of the disease, individual patient parameters including age, physiological state, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dose at which the particles or pharmaceutical compositions described herein are administered can depend on various such parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose via a different, more localized route of administration) can be used.

In certain embodiments, pharmaceutical compositions of the invention (e.g., immunogenic compositions, i.e., pharmaceutical compositions that can be used to induce an immune response) are formulated as a single dose in a container, e.g., a vial. In certain embodiments, the immunogenic compositions are formulated as a multi-dose formulation in a vial. In certain embodiments, the multi-dose formulation contains at least two doses per vial. In certain embodiments, the multi-dose formulation contains a total of 2-20 doses per vial, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In certain embodiments, each dose in a vial is equal in volume. In certain embodiments, the first dose is a different volume from subsequent doses.

A "stable" multi-dose formulation preferably does not exhibit unacceptable levels of microbial growth and is substantially free of or free from degradation or degradation of the active biological molecule components. As used herein, a "stable" immunogenic composition includes a formulation that maintains its ability to elicit a desired immunological response when administered to a subject.

The pharmaceutical compositions of the present invention may include buffers (e.g., from the nucleic acid (e.g., DNA or RNA) composition from which the pharmaceutical composition is prepared), preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions of the present invention, particularly ready-to-use pharmaceutical compositions, include one or more pharmaceutically acceptable carriers, diluents, and/or additives.

Suitable preservatives for use in the pharmaceutical compositions of the present invention include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

As used herein, the term "excipient" refers to a substance that may be present in a pharmaceutical composition of the present invention but is not an active ingredient. Examples of excipients include, but are not limited to, a carrier, binder, diluent, lubricant, thickener, surfactant, preservative, stabilizer, emulsifier, buffer, flavoring agent, or coloring agent.

"Pharmaceutically acceptable salts" include, for example, acid addition salts which may be formed by use of pharmaceutically acceptable acids such as, for example, hydrochloric acid, acetic acid, lactic acid, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), or benzoic acid. Furthermore, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); ammonium (NH ₄ ⁺ ); and salts formed with suitable organic ligands (e.g., quaternary ammonium and amine cations). Illustrative examples of pharmaceutically acceptable salts can be found in the prior art; see, for example, SM Berge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66, pp. 1-19 (1977). Salts that are not pharmaceutically acceptable may be used to prepare pharmaceutically acceptable salts and are included in the present invention.

The term "diluent" refers to a diluting and/or thinning agent. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and/or mixing medium. Examples of suitable diluents include ethanol and water.

The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxy-propylene copolymers.

Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, edit. 1985).

Pharmaceutical carriers, excipients, or diluents may be selected with regard to the intended route of administration and standard pharmaceutical practice.

Routes of Administration of Pharmaceutical Compositions : In certain embodiments, the compositions described herein, e.g., pharmaceutical compositions, particularly ready-to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, cutaneously, intranodal, intramuscularly, or intratumorally. In certain embodiments, the (pharmaceutical) composition is formulated for local or systemic administration. Systemic administration can include enteral administration, including absorption from the digestive tract, or parenteral administration. As used herein, "parenteral administration" refers to any administration other than via the digestive tract, such as intravenous injection. In certain embodiments, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for systemic administration. In certain embodiments, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) composition, particularly the ready-to-use pharmaceutical composition, is formulated for intramuscular administration.

Uses of Pharmaceutical Compositions The nucleic acid (e.g., RNA) compositions described herein may be used for the therapeutic or prophylactic treatment of various diseases, particularly diseases in which delivery of a peptide or protein to a subject provides a therapeutic or prophylactic effect. For example, delivery of an antigen or epitope derived from a virus may be useful for treating or preventing viral diseases caused by the virus. Delivery of a tumor antigen or epitope may be useful for treating cancer diseases in which cancer cells express the tumor antigen. Delivery of a functional protein or enzyme may be useful for treating genetic disorders characterized by protein dysfunction, such as lysosomal storage diseases (e.g., mucopolysaccharidoses) or factor deficiencies. Delivery of cytokines or cytokine-fusions may be useful for controlling the tumor microenvironment.

The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. Disease is often understood as a medical condition associated with specific symptoms and signs. Disease may be caused by factors originating from external sources, such as infectious disease, or by internal malfunctions, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual or causes similar problems in those who come into contact with the individual. This broad meaning may include injury, disability, disorder, syndrome, infection, isolated symptoms, deviant behavior, and atypical variations in structure and function, while in other contexts and for other purposes, these may be considered distinct categories. Disease may affect individuals not only physically but also emotionally, as there are usually many illnesses and living with them can alter one's outlook on life and personality.

The term "antigen-associated disease" refers to any disease in which an antigen is involved, e.g., a disease characterized by the presence of an antigen. The antigen-associated disease can be an infectious disease or a cancer disease, or simply cancer. The antigen can be a disease-associated antigen, e.g., a tumor-associated antigen, a viral antigen, or a bacterial antigen. In some embodiments, the antigen-associated disease is a disease involving cells that express the antigen, preferably presenting the antigen on their cell surface, e.g., in the context of MHC.

The term "infectious disease" refers to any disease that is transmitted from individual to individual or organism to organism and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, viral, bacterial, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases include, for example, sexually transmitted diseases (e.g., chlamydia, gonorrhea, or syphilis), SARS, acquired immune deficiency syndrome (AIDS), measles, chickenpox, cytomegalovirus infection, genital herpes, hepatitis (e.g., hepatitis B or C), influenza (influenza, e.g., human influenza, swine influenza, canine influenza, equine influenza, and avian influenza), HPV infection, shingles, rabies, the common cold, gastroenteritis, rubella, mumps, and anthrax. , cholera, diphtheria, food poisoning, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, whooping cough, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dientamebiasis, giardiasis, amebic dysentery, coccidiosis, toxoplasmosis, sarcocystis, rhinosporidiosis, and balantidiosis.

The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, reproductive organ cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasm, neuroectodermal cancer, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the present invention also includes cancer metastasis.

In some embodiments, the nucleic acid (e.g., DNA or RNA) compositions described herein (including the nucleic acid conjugate compositions described herein) can be used for the therapeutic or prophylactic treatment of infectious diseases.

In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a condition from which a subject is suffering, such as the administration of therapeutically active compounds for the relief of symptoms or complications, delaying the progression of a disease, disorder or condition, reducing or ameliorating symptoms and complications and/or curing or eliminating a disease, disorder or condition, as well as the prevention of a condition, where prevention is understood as the management and care of an individual with the purpose of combating a disease, condition or disorder, and includes the administration of active compounds to prevent the onset of symptoms or complications.

The term "therapeutic treatment" refers to any treatment that improves the health status and/or prolongs (lengthens) the lifespan of an individual. The treatment may eliminate the disease in an individual, halt or slow the progression of the disease in an individual, prevent or slow the progression of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and/or reduce recurrence in an individual who currently has or previously had the disease.

The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to protect an individual from the onset of a disease. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

The terms "individual" and "subject" are used interchangeably herein and refer to a human or other mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) or any other non-mammalian animal, bird (chicken), fish, or any other animal species that may have or be suspected of having a disease or disorder (e.g., cancer, infectious disease), may or may not have the disease or disorder, or may require preventative intervention such as vaccination or intervention such as with protein replacement. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and, therefore, include adults, elderly people, children, and newborns. In embodiments of the present invention, an "individual" or "subject" is a "patient."

The term "patient" refers to an individual or subject for treatment, particularly an affected individual or subject.

In one embodiment of the invention, the goal is to provide protection against infectious diseases through vaccination.

In some embodiments of the present invention, the goal is to deliver nucleic acid (e.g., DNA or RNA) to cells of a subject. Thus, in some embodiments, nucleic acid (e.g., DNA or RNA) can be administered to a subject to induce an immune response by providing a vaccine.

Those skilled in the art will recognize that one of the principles of immunotherapy and vaccination is that an immune protective response against disease is generated by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease being treated. Accordingly, the nucleic acids (e.g., DNA or RNA) described herein can be applied to induce or enhance an immune response. Thus, the nucleic acids described herein are useful for the prophylactic and/or therapeutic treatment of diseases involving the antigen or epitope.

In certain embodiments of the invention, nucleic acids (e.g., DNA or RNA) can be delivered to a subject to deliver a therapeutic or prophylactic peptide or polypeptide (e.g., a pharmaceutically active peptide or polypeptide) to the subject, where the nucleic acid encodes the therapeutic or prophylactic peptide or polypeptide.

In certain embodiments of the invention, a nucleic acid (e.g., DNA or RNA) can be administered to a subject to treat or prevent a disease in the subject, where delivery of the nucleic acid to the subject's cells is beneficial to treat or prevent the disease. In certain embodiments, the nucleic acid encodes a therapeutic or prophylactic peptide or polypeptide, where administration of the therapeutic or prophylactic peptide or polypeptide to a subject is beneficial to treat or prevent the disease.

In certain embodiments of the present invention, the objective is to provide an immune response against diseased cells that express an antigen, such as cancer cells that express a tumor antigen, and to treat diseases, such as cancer diseases, in which cells that express an antigen, such as a tumor antigen, are involved. Thus, in certain embodiments, nucleic acids (e.g., DNA or RNA) can be administered to a subject to provide an immune response against diseased cells that express an antigen, such as cancer cells that express a tumor antigen, and to treat diseases, such as cancer diseases, in which cells that express an antigen, such as a tumor antigen, are involved.

In some embodiments of the present invention, the treatment of cancer by vaccination is of interest. Thus, in some embodiments, nucleic acids (e.g., DNA or RNA) can be administered to a subject for the treatment of cancer by vaccination.

In some embodiments of the present invention, the objective is to provide an immune response against cancer cells that express tumor antigens and to treat cancer diseases involving cells that express tumor antigens. Thus, in some embodiments, nucleic acids (e.g., DNA or RNA) can be administered to a subject to provide an immune response against cancer cells that express tumor antigens and to treat cancer diseases involving cells that express tumor antigens.

In some embodiments of the present invention, the goal is to provide protection against infectious diseases by vaccination. Thus, in some embodiments, nucleic acids (e.g., DNA or RNA) can be administered to a subject to provide protection against infectious diseases by vaccination.

In certain embodiments of the invention, nucleic acids (e.g., DNA or RNA) can be administered to a subject to provide a secreted therapeutic protein, such as an antibody, bispecific antibody, cytokine, cytokine fusion protein, or enzyme, to the subject, particularly a subject in need thereof.

In certain embodiments of the present invention, it is an object to provide a subject, particularly a subject in need thereof, with one or more cytokines or cytokine fusions that modulate the tumor microenvironment. Thus, in certain embodiments, nucleic acids (e.g., DNA or RNA) can be administered to a subject, particularly a subject in need thereof, to provide one or more cytokines or cytokine fusions that modulate the tumor microenvironment.

In certain embodiments of the present invention, it is an objective to provide a subject, particularly a subject in need thereof, with one or more cytokines or cytokine fusions having anti-tumor activity. Thus, in certain embodiments, nucleic acids (e.g., DNA or RNA) can be administered to provide a subject, particularly a subject in need thereof, with one or more cytokines or cytokine fusions having anti-tumor activity.

In certain embodiments of the present invention, nucleic acids (e.g., DNA or RNA) may be administered to a subject, particularly a subject in need thereof, to provide protein replacement therapy, such as for the production of erythropoietin, Factor VII, von Willebrand factor, β-galactosidase, or alpha-N-acetylglucosaminidase.

In some embodiments of the present invention, nucleic acids (e.g., DNA or RNA) may be administered to a subject for modulation/reprogramming of immune cells in the subject's blood.

In certain embodiments of the present invention (particularly those relating to inhibitory RNA), the goal is to reduce or inhibit expression of a peptide or polypeptide (e.g., transcription and/or translation of a target mRNA). Thus, in certain embodiments, nucleic acids (e.g., DNA or RNA) can be administered to reduce or inhibit expression of a peptide or polypeptide (e.g., transcription and/or translation of a target mRNA). In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide, particularly a pharmaceutically active peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with disease. In certain embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (e.g., increased expression compared to expression in healthy subjects) is associated with cancer.

In some embodiments, the nucleic acid is present in a composition described herein.

In some embodiments, the nucleic acid is administered in a pharmaceutically effective amount.

In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human.

Those skilled in the art are aware that one of the principles of immunotherapy and vaccination is that an immune protective response against disease is generated by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease being treated. Accordingly, the pharmaceutical compositions described herein are applicable to inducing or enhancing an immune response. Therefore, the pharmaceutical compositions described herein are useful for the prophylactic and/or therapeutic treatment of diseases involving the antigen or epitope.

The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual for the purpose of inducing an immune response, e.g., for therapeutic or prophylactic reasons.

Citation and examination of any documents referenced herein is not intended as an admission that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the accuracy of the contents of these documents.

This description (including the following examples) is provided to enable one of ordinary skill in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided merely as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the examples described and illustrated herein, but are to be accorded the scope consistent with the appended claims.

Itemized Embodiment 1. A composition comprising: (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) a polymer conjugate compound comprising: (a) a polymer comprising the following general formula (I): and (b) one or more hydrophobic chains:
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100.

2.
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
The composition of item 1.

3. The composition of paragraph 1 or 2, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl.

4. The composition of any of paragraphs 1 to 3, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or methyl.

5. The composition of any of paragraphs 1 to 4, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen.

6. The composition of any one of items 1 to 5, wherein Y is —CH ₂ — or —(CH ₂ ) ₂ —.

7. The composition of any one of paragraphs 1 to 6, wherein Y is —CH ₂ —.

8. The polymer has the following general formula (II):
wherein R ¹ is hydrogen or C _1-8 alkyl.
8. The composition of any one of items 1 to 7, comprising:

9. The composition of any one of items 1 to 8, wherein z is 2 to 10, for example, 2 to 7.

10. The composition of any one of items 1 to 9, wherein z is 2 to 5.

11. The composition of any one of items 1 to 10, wherein z is 2 or 3.

12. The composition of any one of items 1 to 11, wherein z is 2.

13. The polymer has the following general formula (III):
wherein R ¹ is hydrogen or C _1-8 alkyl.
13. The composition of any one of items 1 to 12, comprising:

14. The composition of any of paragraphs 8 to 13, wherein R ¹ is hydrogen or methyl.

15. The composition of any of paragraphs 8 to 14, wherein R ¹ is hydrogen.

16. The polymer has the following general formula (IV):
16. The composition of any one of items 1 to 15, comprising:

17. The composition of any one of items 1 to 16, wherein n is 5 to 50.

18. The composition of any one of items 1 to 17, wherein n is 5 to 25.

19. The composition of any one of items 1 to 18, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

20. The composition of any of paragraphs 1 to 19, wherein one or more hydrophobic chains are located at the ^X1 or ^X2 end of the polymer.

21. The composition of any of paragraphs 1 to 20, wherein one or more hydrophobic chains are acyclic, preferably linear, hydrocarbyl groups, more preferably having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms.

22. The polymer conjugate compound has the following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
22. The composition of any one of items 1 to 21, comprising:

23.
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
Item 23. The composition of item 22.

24. The composition of paragraph 22 or 23, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl.

25. The composition of any of paragraphs 22-24, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or methyl.

26. The composition of any of paragraphs 22-25, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen.

27. The composition of any of paragraphs 22 to 26, wherein Y is —CH ₂ — or —(CH ₂ ) ₂ —.

28. The composition of any of paragraphs 22 to 27, wherein Y is —CH ₂ —.

29. The polymer conjugate compound has the following general formula (VI) or (VI'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
29. The composition of any of items 22 to 28, comprising:

30. The composition of any one of items 22 to 29, wherein z is 2 to 10, for example, 2 to 7.

31. The composition of any one of items 22 to 30, wherein z is 2 to 5.

32. The composition of any one of items 22 to 31, wherein z is 2 or 3.

33. The composition of any one of items 22 to 32, wherein z is 2.

34. A compound represented by the following general formula (VII) or (VII'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
34. The composition of any of items 22 to 33, comprising:

35. The composition of any of paragraphs 29 to 34, wherein R ¹ is hydrogen or methyl.

36. The composition of any of paragraphs 29 to 35, wherein R ¹ is hydrogen.

37. The polymer conjugate compound has the following general formula (VIII) or (VIII'):
37. The composition of any of items 22 to 36, comprising:

38. The composition of any one of items 22 to 37, wherein n is 5 to 50.

39. The composition of any one of items 22 to 38, wherein n is 5 to 25.

40. The composition of any one of items 22 to 39, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

41. The composition of any of paragraphs 22-40, wherein R ² is R ⁴ or -L ¹ (R ⁴ ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group; L ¹ is a linker; and p is 1 or 2.

42. ^L1 comprises at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or the alkylene group is attached at the end attached to ^R4 to a divalent functionalized moiety, wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbohydrate, thioisopropyl ... Nyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal , ketal, hemiketal, imide, and amide moieties; and/or each divalent functionalized moiety is selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionyl a 42. The composition of claim 41, wherein the aryl group is independently selected from an imide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties.

43. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ](C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(=N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N=)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; C _1-6 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁵ is selected from the group consisting of C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); r is an integer from 1 to 2; and 3,4-dihydro-2H-chromen-6-yl is optionally selected from halogen, C _1-3 alkyl, —OH, —CN and —OC 43. The composition of paragraph ₄₁ or 42, wherein the C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to another hydrophobic chain R ⁴ .

44. The composition of any of paragraphs 41 to 43, wherein L ¹ further comprises at least one additional bifunctional moiety through which R ² is attached to X ¹ of formula (V) or X ² of formula (V').

45. At least one further difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide). 45. The composition of paragraph 44, wherein L 1 is selected from the group consisting of , carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties, preferably the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl and thiocarbonyl, wherein when L ¹ further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a C _1-6 -alkylene group.

46. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C 46. The composition of any of items _{41 to} 45, wherein the 3,4 _{-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-3 alkyl} , —OH, —CN and —OC _1-3 alkyl; and the C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to another hydrophobic chain R ⁴ .

47. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) ₂ N-, and [*-C(O)NH](C _1-6 -alkyltriyl)O-, or L ¹ is (*-)(R ²⁶ )N-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R 47. The composition of any of paragraphs 41 to 46, wherein ²⁷ is selected from the group consisting of H and a countercation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to another hydrophobic chain R ⁴ .

48. R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O—, (R ⁴ ) ₂ N—, and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O—, or R ² is (R ⁴ )(R ²⁶ )N—, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl optionally substituted with one or more —OH substituents, and other hydrophobic chains R 48. The composition of any of paragraphs 22 to 47, wherein the compound is directly bonded to ⁴ .

49. The composition of any of paragraphs 22-48, wherein ^R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or ^R2 is a monoalkylamine moiety.

50. The composition of any of paragraphs 41 to 49, wherein each R ⁴ is independently an acyclic, preferably straight-chain, hydrocarbyl group.

51. The composition of any of paragraphs 41 to 50, wherein each R ⁴ is independently a hydrocarbyl group having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms.

52. The composition of any of paragraphs 22-51, wherein ^R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and a palmitoylceramide moiety, or ^R2 is a monomyristylamine moiety.

53. R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ^{21 ,} a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ₂₂ and R 23 are independently selected from the group consisting of H, alkyl, _alkenyl , alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , sugars, amino acids, peptides, and members of a targeting pair; and R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C 53. The composition of any of paragraphs 22 to 52, substituted with one or more substituents independently selected from the group consisting of _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide and a member of a targeting pair.

54. R ³ is selected from the group consisting of H, C _1-3 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein C each of _{the 1-6} alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ and members of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C 54. The composition of any of paragraphs 22 to 53, substituted with one or more substituents independently selected from the group consisting of _(1-3 alkyl) ₂ and members of a targeting pair.

55. The composition of any of paragraphs 22 to 54, wherein R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl), —NH(C _1-3 alkyl), —N(C _1-3 alkyl) ₂ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, _—SH , halogen, _—CN , —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C( _O )NHCH 3 , —C(O)NH(CH 2 ) 2 NH ₂ and members of a targeting pair.

56. The composition of any of paragraphs 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

57. A polymer conjugate compound having the following formula:
[During the ceremony,
n is 5 to 25;
R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl) and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one _or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair;
R ²⁷ is H or a counter cation; and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
57. The composition of any of paragraphs 1 to 56, having one of:

58. The composition of item 57, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

59. The composition of paragraph 57 or 58, wherein R ³ is H or —C(O)(C _1-3 alkyl), wherein the C _1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N ₃ , C _2-6 alkynyl, antigen, or antibody.

60. A polymer conjugate compound having the following formula:
(Wherein, in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case, —C(O)C ₁₅ H ₃₁ refers to the moiety —C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case, —C(O)C ₁₃ H ₂₇ refers to the moiety —C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case, —C ₁₄ H ₂₉ refers to the moiety —(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case, —C ₁₃ H ₂₇ refers to the moiety —(CH ₂ ) ₁₂ CH ₃ , and in each case, n is 8, 10, 12, 14, or 16.)
60. The composition of any of paragraphs 1 to 59, having one of:

61. A polymer conjugate compound having the following formula:
(i)
wherein n is 14 and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl);
(ii)
wherein n is 14 and in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl);
(iii)
wherein n is 8, 12, 14, or 16.
61. The composition of any of claims 1 to 60, comprising one of:

62. The composition of any one of paragraphs 1 to 61, wherein the composition is substantially free of lipids or lipid-like substances containing polyethylene glycol (PEG), wherein the PEG has at least 30 consecutive ethylene glycol repeat units.

63. A composition according to any one of items 1 to 62, wherein water is the main component of the composition and/or the total amount of solvents other than water contained in the composition is less than about 0.5% (v/v).

64. The composition of any one of paragraphs 1 to 63, wherein the concentration of the nucleic acid in the composition is from about 1 mg/L to about 500 mg/L, for example, from about 1 mg/L to about 100 mg/L, from about 5 mg/L to about 100 mg/L, or from about 10 mg/L to about 100 mg/L.

65. The composition of any one of paragraphs 1 to 64, wherein the cationically ionizable lipid comprises a head group containing at least one tertiary amine moiety.

66. A cationic or cationically ionizable lipid of formula (X)
[During the ceremony,
one of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)—, —C(═O)NR ^a —, NR ^a C(═O)NR ^a —, —OC(═O)NR ^a — or —NR ^a C(═O)O—; and the other of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)NR ^a -, NR ^a C(═O)NR ^a -, -OC(═O)NR ^a - or -NR ^a C(═O)O- or a direct bond;
G ¹ and G ² are each independently unsubstituted C ₁ -C ₁₂ alkylene or C _2-12 alkenylene;
G ³ is C _1-24 alkylene, C _2-24 alkenylene, C _3-8 cycloalkylene or C _3-8 cycloalkenylene;
R ^a is H or C _1-12 alkyl;
R ³⁵ and R ³⁶ are each independently C _6-24 alkyl or C _6-24 alkenyl;
R ³⁷ is H, OR ⁵⁰ , CN, —C(═O)OR ⁴⁰ , —OC(═O)R ⁴⁰ or —NR ⁵⁰ C(═O)R ⁴⁰ ;
R ⁴⁰ is C _1-12 alkyl;
R ⁵⁰ is H or C _1-6 alkyl; and x is 0, 1, or 2.
66. The composition of any one of items 1 to 65, having the structure of: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

67. A cationic or cationically ionizable lipid having the formula (XI):
[During the ceremony,
each of R ₁ and R ₂ is independently R ₅ or -G ₁ -L ₁ -R ₆ , where at least one of R ₁ and R ₂ is -G ₁ -L ₁ -R ₆ ;
each of R ₃ and R ₄ is independently selected from the group consisting of C _1-6 alkyl, C _2-6 alkenyl, aryl, and C _3-10 cycloalkyl;
_R5 and _R6 are each independently an acyclic hydrocarbyl group having at least 10 carbon atoms;
each of G ₁ and G ₂ independently is unsubstituted C _1-12 alkylene or C _2-12 alkenylene;
each of L ₁ and L ₂ is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, —NRaC(═O)NRa—, —OC(═O)NRa— and —NRaC(═O)O—;
Ra is H or C _1-12 alkyl;
m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2.
66. The composition of any of items 1 to 65, having the structure:

68. Cationic or cationic ionizable lipids include 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), and N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP). Any of the compositions of items 1 to 65, comprising ammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof.

69. The composition of any one of paragraphs 1 to 68, wherein the cationic or cationically ionizable lipid comprises from about 20 mol% to about 80 mol% of the total lipid present in the composition.

70. The composition of any one of items 1 to 69, further comprising one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of phospholipids and steroids.

71. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 The composition of item 70, wherein the oleoylphosphatidylethanolamine is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE).

72. The composition of item 70 or 71, comprising from about 5 mol% to about 30 mol% of the total lipids present in the phospholipid composition.

73. The composition of any one of paragraphs 70 to 72, wherein the steroid comprises a sterol such as cholesterol.

74. The composition of any one of paragraphs 70 to 73, wherein the steroid constitutes about 10 mol% to about 60 mol% of the total lipids present in the composition.

75. The composition of any of paragraphs 70 to 74, wherein the cationic or cationically ionizable lipid constitutes about 20 mol% to about 70 mol% of the total lipid present in the composition; the polymer conjugate compound constitutes about 0.5 mol% to about 15 mol% of the total lipid present in the composition; the phospholipid constitutes about 5 mol% to about 25 mol% of the total lipid present in the composition; and the steroid constitutes about 20 mol% to about 55 mol% of the total lipid present in the composition.

76. The composition of any one of paragraphs 1 to 75, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationic or cationically ionizable lipid, and at least a portion of a polymer-conjugated compound.

77. The composition of claim 76, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof.

78. The composition of paragraph 76 or 77, wherein the particles constitute at least 50%, preferably at least 75%, and more preferably at least 85% of the nucleic acids present in the composition.

79. The composition of any one of paragraphs 76 to 78, wherein the particles have a size of about 30 nm to about 500 nm.

80. The composition of any one of paragraphs 1 to 79, wherein the nucleic acid is RNA, preferably mRNA.

81. The composition of paragraph 80, wherein the RNA (1) comprises modified nucleosides in place of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (2) has a codon-optimized coding sequence; and/or (3) has a coding sequence with an increased G/C content compared to the wild-type coding sequence.

82. The composition of paragraph 80 or 81, wherein the RNA comprises at least one, preferably all, of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence.

83. The composition of paragraph 82, wherein the polyA sequence comprises at least 100 A nucleotides, and wherein the polyA sequence is preferably an interrupted sequence of A nucleotides.

84. The composition of item 82 or 83, wherein the 5' cap is a cap 1 or cap 2 structure.

85. The composition of any of paragraphs 80 to 84, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and/or contain an epitope for inducing an immune response against an antigen in a subject.

86. The composition of paragraph 85, wherein the pharmaceutically active polypeptide and/or antigen or epitope is derived from or is a pathogen protein, an immunogenic variant of the protein, or an immunogenic fragment of the protein or an immunogenic variant thereof.

87. A method for delivering a nucleic acid to a cell of a subject, comprising administering to the subject a composition according to any one of paragraphs 1 to 86.

88. A method for delivering a therapeutic peptide or protein to a subject, comprising administering to the subject the composition of any one of paragraphs 1 to 86, wherein the nucleic acid encodes the therapeutic peptide or protein.

89. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition according to any one of paragraphs 1 to 86, wherein delivery of the nucleic acid to the subject's cells is beneficial to the treatment or prevention of the disease or disorder.

90. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of any one of paragraphs 1 to 86, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

91. The method of any one of paragraphs 87 to 90, wherein the subject is a mammal.

92. The method of paragraph 91, wherein the mammal is a human.

93. (a) A compound of the following general formula (I):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100.
and (b) one or more hydrophobic chains.

94.
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
94. The polymer conjugate compound of claim 93.

95. The polymer conjugate compound of paragraph 93 or 94, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl.

96. The polymer conjugate compound of any of paragraphs 93 to 95, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or methyl.

97. The polymer conjugate compound of any of paragraphs 93 to 96, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen.

98. The polymer conjugate compound of any of paragraphs 93 to 97, wherein Y is —CH ₂ — or —(CH ₂ ) ₂ —.

99. The polymer conjugate compound of any of paragraphs 93 to 98, wherein Y is —CH ₂ —.

100. A polymer having the following general formula (II):
wherein R ¹ is hydrogen or C _1-8 alkyl.
99. The polymer conjugate compound of any of paragraphs 93 to 99, comprising:

101. The polymer conjugate compound of any of items 93 to 100, wherein z is 2 to 10, for example, 2 to 7.

102. The polymer conjugate compound of any one of items 93 to 101, wherein z is 2 to 5.

103. The polymer conjugate compound of any one of items 93 to 102, wherein z is 2 or 3.

104. The polymer conjugate compound of any one of items 93 to 103, wherein z is 2.

105. A polymer having the following general formula (III):
wherein R ¹ is hydrogen or C _1-8 alkyl.
105. The polymer conjugate compound of any of paragraphs 93 to 104, comprising:

106. The polymer conjugate compound of any of paragraphs 100 to 105, wherein R ¹ is hydrogen or methyl.

107. The polymer conjugate compound of any of paragraphs 100 to 106, wherein R ¹ is hydrogen.

108. A polymer having the following general formula (IV):
108. The polymer conjugate compound of any of paragraphs 93 to 107, comprising:

109. The polymer conjugate compound of any one of items 93 to 108, wherein n is 5 to 50.

110. The polymer conjugate compound of any one of items 93 to 109, wherein n is 5 to 25.

111. The polymer conjugate compound of any one of items 93 to 110, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

112. The polymer conjugate compound of any of paragraphs 93 to 111, wherein one or more hydrophobic chains are located at the ^X1 or ^X2 end of the polymer.

113. The polymer conjugate compound of any of paragraphs 93 to 112, wherein one or more hydrophobic chains are acyclic, preferably linear, hydrocarbyl groups, more preferably having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms.

114. A compound represented by the following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
114. The polymer conjugate compound of any of paragraphs 93 to 113, comprising:

115.
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
116. The polymer conjugate compound of paragraph 114 or ₁₁₅ , wherein X ¹ is —C(O)— and X ² is —NR ¹ —, wherein R ¹ is hydrogen or C _1-8 alkyl. The polymer conjugate compound of paragraph 114, wherein X ¹ is —C(O)— and X 2 is —NR 1 —, wherein R 1 is hydrogen or C 1-8 alkyl.

117. The polymer conjugate compound of any of paragraphs 114 to 116, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or methyl.

118. The polymer conjugate compound of any of paragraphs 114 to 117, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen.

119. The polymer conjugate compound of any of paragraphs 114 to 118, wherein Y is —CH ₂ — or —(CH ₂ ) ₂ —.

120. The polymer conjugate compound of any of paragraphs 114 to 119, wherein Y is —CH ₂ —.

121. A compound represented by the following general formula (VI) or (VI'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
1202. The polymer conjugate compound of any of paragraphs 114 to 1201, comprising:

122. The polymer conjugate compound of any of items 114 to 121, wherein z is 2 to 10, for example, 2 to 7.

123. The polymer conjugate compound of any one of items 114 to 122, wherein z is 2 to 5.

124. The polymer conjugate compound of any one of items 114 to 123, wherein z is 2 or 3.

125. The polymer conjugate compound of any one of items 114 to 124, wherein z is 2.

126. A compound represented by the following general formula (VII) or (VII'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
126. The polymer conjugate compound of any of paragraphs 114 to 125, comprising:

127. The polymer conjugate compound of any of paragraphs 121 to 126, wherein R ¹ is hydrogen or methyl.

128. The polymer conjugate compound of any of paragraphs 121 to 127, wherein R ¹ is hydrogen.

129. A compound represented by the following general formula (VIII) or (VIII'):
129. The polymer conjugate compound of any of paragraphs 114 to 128, comprising:

130. The polymer conjugate compound of any one of items 114 to 129, wherein n is 5 to 50.

131. The polymer conjugate compound of any one of items 114 to 130, wherein n is 5 to 25.

132. The polymer conjugate compound of any one of items 114 to 131, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

133. The polymer conjugate compound of any of paragraphs 114 to 132, wherein R ² is R ⁴ or -L ¹ (R ⁴ ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group; L ¹ is a linker; and p is 1 or 2.

134. L ¹ comprises at least one functionalized moiety, such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or the alkylene group is attached to a divalent functionalized moiety at the terminus attached to R ⁴ , wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, Orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, and/or each divalent functionalized moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate 134. The polymer conjugate compound of claim 133, wherein the aryl group is independently selected from a aryl group, a carbonothioate, a carbonodithioate, a carbonotrithioate, a guanidino(imidamide), a carbamimidate, a carbonimidate, a carbamate, a carbamodithioate, a carbonodithioimidate, a carbamimidothioate, a carbamothioate, a carbonimidothioate, an acylhydrazone, a hydrazine, an oxime, an acetal, a hemiacetal, a ketal, a hemiketal, an imine, an imide, and an amide moiety.

135. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ](C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(=N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N=)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; C _1-6 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁵ is selected from the group consisting of C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); r is an integer from 1 to 2; and 3,4-dihydro-2H-chromen-6-yl is optionally selected from halogen, C _1-3 alkyl, —OH, —CN and —OC 135. The polymer conjugate compound of paragraph ₁₃₃ or 134, wherein the C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly attached to another hydrophobic chain R ⁴ .

136. The polymer conjugate compound of any of paragraphs 133 to 135, wherein L ¹ further comprises at least one additional bifunctionalized moiety through which R ² is attached to X ¹ of formula (V) or X ² of formula (V').

137. At least one further difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imide) amide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties, preferably the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl and thiocarbonyl, wherein when L ¹ further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a C _1-6 -alkylene group.

138. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C 138. The polymer conjugate compound of any of paragraphs ₁₃₃ to 137, wherein 3,4-dihydro- _{2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-3 alkyl} , —OH, —CN and —OC _1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ .

139. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) ₂ N-, and [*-C(O)NH](C _1-6 -alkyltriyl)O-, or L ¹ is (*-)(R ²⁶ )N-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R 139. The polymer conjugate compound of any of paragraphs ₁₃₃ to ₁₃₈ , wherein ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-3 alkyl, —OH, —CN and —OC 1-3 alkyl; and C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents, and is directly attached to another hydrophobic chain R ⁴ .

140. R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O—(C _1-3 -alkylene), [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O—, (R ⁴ ) ₂ N—, and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O—, or R ² is (R ⁴ )(R ²⁶ )N—, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl optionally substituted with one or more —OH substituents, and other hydrophobic chains R 140. The polymer conjugate compound of any of paragraphs 114 to 139, which is directly bonded to ⁴ .

141. The polymer conjugate compound of any of paragraphs 114-140, wherein R ² is selected from phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or R ² is a monoalkylamine moiety.

142. The polymer conjugate compound of any of paragraphs 133 to 141, wherein each R ⁴ is independently an acyclic, preferably straight-chain, hydrocarbyl group.

143. The polymer conjugate compound of any of paragraphs 133 to 142, wherein each R ⁴ is independently a hydrocarbyl group having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms.

144. The polymer conjugate compound of any of paragraphs 114-143, wherein ^R2 is selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or ^R2 is a monomyristylamine moiety.

145. R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ^{21 ,} a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ₂₂ and R 23 are independently selected from the group consisting of H, alkyl, _alkenyl , alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , sugars, amino acids, peptides, and members of a targeting pair; and R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C 145. The polymer conjugate compound of any of paragraphs 114 to 144, substituted with one or more substituents independently selected from the group consisting of _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide and a member of a targeting pair.

146. R ³ is selected from the group consisting of H, C _1-3 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein C each of _{the 1-6} alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ and members of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C 146. The polymer conjugate compound of any of paragraphs 114 to 145, substituted with one or more substituents independently selected from the group consisting of _(1-3 alkyl) ₂ and members of a targeting pair.

147. The polymer conjugate compound of any of paragraphs 114 to 146, wherein R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl), —NH(C _1-3 alkyl) and —N(C _1-3 alkyl) ₂ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or _more substituents independently selected from the group consisting of _—OH , —SH, halogen, _—CN , —N ₃ , C _2-6 alkynyl, —COOH, —COOCH 3 , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH 2 , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair.

148. The polymer conjugate compound of any of paragraphs 114 to 147, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor.

149. The following formula:
[During the ceremony,
n is 5 to 25;
R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl) and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one _or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair;
R ²⁷ is H or a counter cation; and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
149. The polymer conjugate compound of any of paragraphs 93 to 148, having the formula:

150. The polymer conjugate compound of item 149, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16.

151. The polymer conjugate compound of paragraph 149 or ₁₅₀ , wherein R ³ is H or —C(O)(C _1-3 alkyl), wherein the C _1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N _3, and C 2-6 alkynyl.

152. The following formula:
(Wherein, in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case, —C(O)C ₁₅ H ₃₁ refers to the moiety —C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case, —C(O)C ₁₃ H ₂₇ refers to the moiety —C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case, —C ₁₄ H ₂₉ refers to the moiety —(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case, —C ₁₃ H ₂₇ refers to the moiety —(CH ₂ ) ₁₂ CH ₃ , and in each case, n is 8, 10, 12, or 14.)
152. The polymer conjugate compound of any of paragraphs 93 to 151, having the formula:

153. The following formula:
(i)
wherein n is 14 and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl);
(ii)
wherein n is 14 and in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl);
(iii)
wherein n is 8, 12, 14, or 16.
153. The polymer conjugate compound of any of paragraphs 93 to 152, having the formula:

154. (a) A polymer conjugate compound of any of items 93 to 153 comprising a member of a targeting pair; and (b) a conjugate of a compound comprising another member of the targeting pair.

155. The conjugate of paragraph 154, wherein the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (e.g., an antigen or epitope), or an antibody.

156. The following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); m1 and m2 are each independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
156. A conjugate of paragraph 154 or 155, which has one of:

157. The following expression
(i)
wherein m1 is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2;
(ii)
[In the formula, m1 and m2 each independently represent 1, 2, or 3, and preferably m1 is 1 and m2 is 2, or m1 is 2 and m2 is 1.]
157. The conjugate of paragraph 156, having one of:

158. The following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
158. A conjugate according to any of paragraphs 154 to 157, which has one of the following groups:

159. A polymer conjugate compound comprising a member of a targeting pair having the following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
having
155. The conjugate of claim 154, wherein the compound comprising the other member of the targeting pair is (i) an antibody specific for the antigen if Pept is the antigen; or (ii) a compound comprising the antigen if Pept is an antibody specific for the antigen; and the polymer conjugate compound comprising a member of the targeting pair is conjugated to the compound comprising the other member of the targeting pair via the interaction of (1) the antibody specific for the antigen and (2) the antigen.

Further aspects of the present invention are disclosed herein.

Materials: Cholesterol and distearoyl-sn-glycerol-3-phosphocholine (DSPC) were supplied by Avanti (Alabama, USA). The cationically ionizable lipid HY-501 (also referred to herein as the cationically ionizable lipid of formula XIV-3) was purchased from NucleoSyn (Olivet, France). Absolute ethanol, citrate buffer, and OmniPur® water (WFI quality, sterile purified water) were purchased from Merck Millipore. RNase-free TE buffer concentrate, 20x, and RiboGreen dye used in the RiboGreen® RNA quantification assay were purchased from Invitrogen. Triton X-100 was purchased from VWR International GmbH, Darmstadt, Germany. (+)-α-Tocopherol and DMA (dimyristylamine) were purchased from Sigma-Aldrich, Inc. DMG (1,2-dimyristoylglycerol) was purchased from Biosynth International, Inc. Fmoc-AEEA-OH was purchased from Ambeed Inc. DSPE was purchased from Echelon Biosciences, Inc. All materials in contact with the solutions and LNP preparations were sterile. V09 mRNA (modified mRNA encoding the P2 S protein of SARS-CoV-2) used in LNP experiments was synthesized in-house using an internal protocol. Alternatively, luciferase mRNA (LUC mRNA, a modified mRNA encoding firefly luciferase) or Thy1.1 RNA (encoding thymocyte antigen 1.1) was used.

Methods All methods, including kit and reagent specifications, are performed according to manufacturer's instructions unless otherwise noted.

Salt Exchange and Purification of Amphiphiles: The lyophilized composition containing the amphiphile to be purified is resuspended in ammonium acetate (10 g/L) in 90% water, 10% acetonitrile and loaded onto a preparative HPLC column (XBridge Protein BEH C ₄ OBD Prep column, 300 Å, 5 μm, 10 mm x 50 mm) using an autoinjector via a 10 mL sample loop. Sample loading is performed with ammonium acetate (10 g/L) in 90% water, 10% acetonitrile, followed by a 15-minute wash through the sample. Mobile phase A is then switched to 1% TFA in water (or 1% acetic acid) and run through the column for 15 minutes, followed by a 0.1-minute jump to 95% B (acetonitrile). Fraction elution is triggered (or by mass spectrometry) with the same mass used in the purification step. The eluted fractions are pooled together. An aliquot is injected into the UPLC-MS for purity and ID. The pooled samples are lyophilized to dryness.

For certain amphiphilic compounds (e.g., those containing a DSPE moiety), the following adapted method can be used: The lyophilized composition containing the amphiphilic compound to be purified is resuspended in ammonium acetate (10 g/L) in 90% water, 10% acetonitrile and loaded onto a preparative HPLC column (XBridge Protein BEH C ₄ OBD Prep column, 300 Å, 5 μm, 10 mm × 50 mm) with an autoinjector via a 10 mL sample loop. Sample loading is performed with ammonium acetate (10 g/L) in 90% water, 10% acetonitrile, with a 15-minute wash through the sample. Mobile phase A is then switched to 95% water, 5% acetonitrile + 1% acetic acid, and mobile phase B is 100% acetonitrile. Due to the hydrophobicity of the amphiphilic compound, a steep gradient for elution is used, as shown in the following table:

Using this gradient, for example, Ac-(AEEA) ₁₄ -DSPE elutes between 60 and 95% B, while more hydrophilic impurities elute earlier. Due to the steepness of the elution gradient, closely eluting impurities may be collected along with the main peak. Therefore, the DPSE starting lipid must be completely consumed during the reaction. Fractions are collected by MS using the M+2, M+3, and M+4 values of each compound; however, absorbance at 214 nm can be used for collection as well. Fractions with estimated or measured UV 214 purity (preferably >95%) are collected in polypropylene vials and lyophilized. They are then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid, transferred to glass vials, and lyophilized for at least two days. Fraction and final QC are performed according to the methods described in the "Determination of Amphiphilic Compound Purity and Identity" section below.

If another purification solvent for the amphiphile (e.g., chloroform) is still present in the composition containing the amphiphile to be purified, it is removed (e.g., using a Biotage V10 rotary evaporator) and the sample is resuspended in methanol. Purification may be performed using reverse-phase HPLC. The preparative column is a Waters XBridge Protein BEH C ₄ OBD Prep column, 300 Å, 5 μm, 19 mm x 100 mm. Mobile phase A is 1% acetic acid in water and mobile phase B is 100% acetonitrile. The flow rate is 15 mL/min. Exemplary values for the gradient and elution points of certain amphiphiles are shown in the following table.

Fractions are collected by MS using the M+2, M+3, and M+4 values of each compound; however, absorbance at 214 nm can be used for collection as well. Fractions with estimated or measured UV214 purity are collected in polypropylene vials and lyophilized. They are then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid, transferred to glass vials, and lyophilized for at least two days. Fraction and final QC are performed according to the methods described in the "Determination of Amphiphilic Compound Purity and Identity" section below.

If the additional amphiphile purification solvent is still present in the composition containing the amphiphile to be purified, it is removed (e.g., using a Biotage V10 rotary evaporator) and the sample is resuspended in chloroform. Purification can be performed using size-exclusion HPLC. The preparative column series is a JAIGEL 2.5 HR and a JAIGEL 2HR. The mobile phase is 100% chloroform or, if necessary for solubility, 90% methanol, 10% chloroform. The flow rate is 10 mL/min. Recycling can be performed to resolve impurities. Fractions are collected by UV absorption. Purity-relevant fractions are pooled and the solvent is removed (e.g., using rotary evaporation). The amphiphile is then resuspended in 50/50 water/acetonitrile + 0.5% acetic acid and lyophilized for at least two days. Fraction and final QC are performed according to the methods described in the "Determination of Amphiphile Purity and Identity" section.

Where applicable, co-eluting hydrophilic impurities from preparative size-exclusion HPLC can be removed by aqueous extraction. The sample is resuspended in chloroform, water is added, and the sample is vortexed. The aqueous layer is separated from the organic layer, and the aqueous layer containing the hydrophilic impurities is removed. The chloroform is then removed from the organic layer (e.g., using a Biotage V10 rotary evaporator). The sample is resuspended in 50/50 water/acetonitrile + 0.5% acetic acid and lyophilized for at least two days.

Determination of Amphiphile Purity and Identity. The purity and identity of the amphiphiles were determined on a Waters H-Class UPLC/MS system containing a Waters H-Class UPLC equipped with a PDA UV detector and a QDa mass detector using a linear gradient of 10-80% B in 8 min at a flow rate of 0.5 mL/min, where mobile phase A was 0.100% TFA in water and mobile phase B was 0.085% TFA in acetonitrile. The column (ACQUITY UPLC PROTEIN BEH C4 column, 300 Å, 1.7 μm, 2.1 mm x 100 mm) was heated to 60°C. The following table shows the parameters used in this analysis. Integration was performed automatically in Empower 3 software.

The acceptance criteria for the final conjugate are 95% UV214 purity and a molecular weight (MW) within 1 amu of the theoretical value.

UPLC-MS
A crude sample (e.g., crude peptide) is dissolved in 50/50 water/acetonitrile + 0.05% TFA to approximately 30 mg/mL. The solution is vortexed and sonicated until completely dissolved (no visible solid material is present). The solution is loaded onto a preparative HPLC column (Phenomenex Luna C18(2) LC column, 100 Å pore size, 10 μm particle size, 30 mm x 250 mm) with an autoinjector via a 10 mL sample loop. The sump load is 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Peptide elution is performed with a 36-minute linear gradient from 20 to 35% B. The flow rate is 30 mL/min. A Waters SQD2 mass spectrometer is used to automatically trigger fraction acquisition based on the M+1H, M+2H, and M+3H values of the target compound (e.g., target peptide). UV ₂₁₄ absorbance is monitored but not used for fraction triggering. Aliquots of selected compound fractions are injected onto a Waters H-class UPLC/MS system using a linear gradient of 10 to 80% B in 8 minutes at a flow rate of 0.5 mL/min, where mobile phase A is 0.100% TFA in water and mobile phase B is 0.085% TFA in acetonitrile. The column (ACQUITY UPLC PEPTIDE BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 100 mm) is heated to 40°C. For some compounds (e.g., peptides), in-process purity measurements are eliminated by visual monitoring of preparative HPLC UV ₂₁₄ absorbance and MS to estimate which fractions are highly pure. Fractions measured or estimated to be ≥ 90% pure are combined into a tared polypropylene tube and lyophilized. If the volume of pure fractions exceeds the tube capacity, multiple polypropylene tubes are filled and lyophilized. Each compound (e.g., peptide) is then resuspended in 50/50 water/acetonitrile + 0.05% TFA, combined into a single tared polypropylene tube, and lyophilized to dryness. Approximately 1 mg of peptide is dissolved to 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected into the UPLC/MS for purity and MW.

Resin Loading of Peptide onto 2-Cltrt Polystyrene Resin: Weigh 1 g of peptide (e.g., 2.59 mmol of Fmoc-AEEA-OH) into a 50 ml conical tube and dissolve in enough DCM to allow for complete dissolution of the amino acid material and sufficient volume to provide resin coverage and mobility during loading. To provide ideal resin loading substitution, double the millimolar amount of peptide (e.g., 5.19 mmol for Fmoc-AEEA-OH) and use this to calculate the amount of 2-cltrt resin required (e.g., the final amount of 2-cltrt resin used to load Fmoc-AEEA-OH was approximately 4.63 g). Once the required amount of resin has been calculated and weighed into two 20 ml syringes, add enough DCM to pre-swell the resin in preparation for loading. Resin pre-swelling continues for approximately 30 minutes to 1 hour, and after that time is complete, 10 equivalents (eq.) of neat DIEA are added to the amino acid solution. Once the DIEA is added, it is added to the resin syringe, which serves as a solvent for transferring the amino acid solution to a 250 ml flat-bottom centrifuge bottle. The 250 ml centrifuge bottle is capped and secured on an orbital shaker for a reaction time of a minimum of 2 hours to a maximum of overnight. Once the reaction time is complete, the resin and amino acid solution are filtered back into the 20 ml syringe used for swelling, and the resin is rinsed 3-5 times with DCM to remove any excess amino acid solution. After rinsing is complete, a solution containing a mixture ratio of 17:2:1 (DCM:methanol:DIEA) is added to each syringe to cap any unreacted sites on the resin. This capping reaction is carried out twice, for 15 minutes each. Once the second capping reaction is complete, rinse the resin five times with DCM and dry in a vacuum manifold or vacuum desiccator for 30 minutes to 1 hour.

Synthesis of Peptide Intermediates (eg, Ac-(AEEA) ₁₄ -OH or Ac-(AEEA) ₈ -OH) Several exemplary protocols are described for the synthesis of peptide intermediates:
Protocol 1
Synthesis of peptide intermediates (e.g., Ac-(AEEA) ₁₄ -OH) can be carried out on a 0.1 mmol scale using a Liberty Blue HT24 synthesizer. Pre-loaded 2-cltrt resin from the above method, "Resin Loading of Peptides onto 2-Cltrt Polystyrene Resin," is added to a Liberty HT suspended in 10 ml of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The synthesis can involve multiple iterations of the loaded material on a 0.1 mmol scale. For example, the synthesis of Ac-(AEEA) ₁₄ -OH or Ac-(AEEA) ₈ -OH can be as follows: The synthesis began with deprotection of the N-terminal α-Fmoc protecting group of Fmoc-(AEEA)-OH using 4 mL of 20% piperidine in DMF by heating at 60 °C for 3 minutes in a microwave with nitrogen sparging every 3 seconds for mixing. After draining, the resin was washed three times with 5 mL of DMF, for 5 seconds each. For the coupling reaction, 2.5 mL of a 0.3 M amino acid solution of Fmoc-AEEA-OH in DMF (7.5 equiv.), 1 mL of 1 M DIC (10 equiv.), and 0.5 mL of 1 M Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 equiv.) was then added to the reaction vessel (RV). Single or double coupling steps were performed by microwave heating at 50 °C for 10 minutes with nitrogen sparging every 3 seconds for mixing during the coupling cycle. After the coupling time is complete, the resin is drained, and 20% piperidine in DMF is added to the reaction vessel (RV) to initiate deprotection (preferably at 60°C) in preparation for the next incoming amino acid. This cycle of Fmoc removal and coupling is repeated sequentially for all amino acids, preferably terminating with a final Fmoc deprotection cycle to provide a free N-terminus for acetyl capping (final deprotection of the amino acid sequence is not performed for the Fmoc-(AEEA) ₁₄ -OH linker). After the instrument run is complete, the resin is transferred to 24 ml fritted syringes using DMF. Approximately 10 ml of capping solution containing N-methylpyrrolidone (NMP), 6.25% acetic anhydride, and 12.5% 2 M diisopropylethylamine (DIEA) in 81.25% DMF is added to each syringe. The capping reaction is allowed to proceed for 15 minutes at room temperature (RT). After 15 minutes, the capping solution is removed from the syringe, the resin is rinsed once with DMF, and the capping reaction is repeated for another 15 minutes. After the second capping reaction is complete, the resin is rinsed five times with 5 ml DMF, then five times with 5 ml DCM in preparation for cleavage of the resin.

Prepare a cleavage solution containing 92.5% trifluoroacetic acid (TFA), 3.75% water, and 3.75% triisopropylsilane (TIPS). Add 10 mL of cleavage solution to each syringe (for peptides prepared on a 0.1 mmol scale). Cleave the peptides on a shaker for at least 2 hours at room temperature. The cleaved peptides are then expelled into the syringes. We have found it efficient to combine all peptides into one large 250 mL bottle so they can be evaporated simultaneously under nitrogen. pAEE peptides do not precipitate in solutions using ether or hexane because the cleavage solution is evaporated under nitrogen in a fume hood (air or rotary evaporation could potentially be used instead of nitrogen). After evaporation, the 250 mL bottle typically contains a gel-like mass of crude peptide material. This is resuspended in enough 50/50 water/acetonitrile + 0.05% TFA so that each repetition can be returned to each conical tube containing approximately 10 mL of resuspension solution.

Purification of peptide intermediates (e.g., Ac-(AEEA) ₁₄ -OH or Ac-(AEEA) ₈ -OH): After cleavage from the resin, each synthetic replicate is resuspended in 50/50 water/acetonitrile + 0.05% TFA, frozen, and lyophilized for 1-2 days. After lyophilization, samples are dissolved to approximately 50 mg/mL in 50/50 water/acetonitrile + 0.05% TFA. The solution is vortexed and sonicated until completely dissolved (no visible solid material is present). An aliquot is taken for crude QC (see the "UPLC-MS Analytical QC Method for Peptide Intermediates" section below). Purification is performed on a Waters Autopurification System equipped with an SQD2 mass detector. The sample is loaded onto a preparative HPLC column (Phenomenex Luna C18(2) LC column, 100 Å pore size, 10 μm particle size) with an autoinjector via a 10 mL sample loop. Sump loading is performed with 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Elution can be performed with a 36-minute linear gradient (duration adapted to scale, if desired, e.g., using a 42-minute linear gradient on the 0.3 mmol scale). Column size, flow rate, and gradient can also be adapted to scale. For example, the following table provides an exemplary purification gradient for the peptide intermediates Ac-(AEEA) ₁₄ -OH and Ac-(AEEA) ₈ -OH.

A Waters SQD2 mass spectrometer is used to automatically trigger fraction collection based on the M+1H, M+2H, and M+3H values of the target peptide. UV214 absorbance is monitored but not used for fraction triggering. Aliquots of selected fractions are subjected to the QC method described in the "HPLC-MS Analytical QC Method for Peptide Intermediates" section. For some fractions, in-process purity measurements can be eliminated by visually monitoring preparative HPLC-MS data to estimate which fractions are highly pure. Fractions with measured or estimated purity ≥ 90% are combined into tared polypropylene tubes and lyophilized. If the volume of pure fractions exceeds the tube capacity, multiple polypropylene tubes are filled and lyophilized. Each peptide is then resuspended in 50/50 water/acetonitrile + 0.05% TFA, combined into a single tared polypropylene tube, and lyophilized for at least two days. An aliquot will be taken from the combined pool and diluted with 50/50 water/acetonitrile + 0.05% TFA for QC.

Protocol 2
The synthesis of peptide intermediates (e.g., Ac-(AEEA) ₁₄ -OH) can be carried out on a 36.6 mmol scale using semi-automated or automated synthesizers without the aid of a microwave. The 2-cltrt resin is initially loaded with Fmoc-AEEA-OH using PyBOP and DIPEA for 3 hours at room temperature. The synthesis can involve multiple replicates on a 0.2 mmol scale for the added material. The synthesis of peptide intermediates can be as follows: deprotection of the α Fmoc protecting group of the resin-bound Fmoc-(AEEA)-OH residue, followed by coupling of the next Fmoc-AEEA-OH until the desired sequence is reached. Deprotection of the Fmoc protecting group is carried out twice for 15 minutes using 30% piperidine in DMF. After draining the Fmoc deprotection solution, the resin is washed with DMF. The next coupling with Fmoc-AEEA-OH is then carried out using PyBOP and DIPEA for 3 hours at room temperature. After the coupling time is reached, the resin is drained and 20% piperidine in DMF is added to the reaction vessel (RV) for the next coupling cycle. After all coupling/deprotection cycles leading to a fully assembled sequence are completed, a final Fmoc deprotection is carried out to provide a free N-terminus for acetyl capping. The free N-terminal amine is then acetylated at room temperature. After capping, the solution is removed from the resin and rinsed with DMF. The syringe and resin are rinsed with DMF. A cleavage solution for removing the peptide from the resin is prepared containing 25% HFIP in DCM for 1 hour at room temperature. The cleaved peptide is then expelled into a syringe, evaporated, and injected for purification.

Purification of the Peptide Intermediate Ac-(AEEA) ₁₄ -OH. After cleavage from the resin, the crude peptide is solubilized and purified by RP-HPLC using an Agilent PLRP-S 100 Å, 50 x 300 mm, 8 μm, 50 x 300 mm system. Elution is performed with eluent A (1% AcOH in H ₂ O) and eluent B (ACN), linearly from 0 to 45% B in 140 min. This choice of eluent allows for the combination of purification and salt exchange in the same process step. Fractions showing a purity ≥ 90% are combined and lyophilized.

UPLC-MS Analytical QC Method for Peptide Intermediates (e.g., Ac-(AEEA) ₁₄ -OH or Ac-(AEEA) ₈ -OH) Purity and identity of peptide intermediates are performed by UPLC-UV-MS using a Waters H-Class UPLC equipped with a PDA UV detector and a QDa mass detector. The following table shows the UPLC parameters used for this analysis. Integration is performed automatically in Empower 3 software.

Analysis method 1
Acceptance criteria for peptide intermediates are 90% UV214 purity and MW within 1 amu of theoretical.

Analysis method 2

Anti-PEG ELISA Assay of Polyclonal Anti-PEG Antibody Binding This assay was used to determine the binding of anti-PEG polyclonal antibodies to various antigens (PEG, AEEA, pSAR).

reagent:
Pierce Neutravidin High Binding Plate, 8-well Strips, 15508
Assay buffer: 1x DPBS, 2% BSA, 0.1% CHAPS
・Washing buffer: 1x DPBS, 0.1% CHAPS
Rabbit polyclonal anti-PEG, Life diagnostics #PEGPAB-01, 1 mg/mL
Rabbit polyclonal anti-PEG, cloud-clone corp #PAX163Ge01, 1 mg/mL
Goat anti-rabbit IgG (H+L) secondary antibody, HRP, Thermo A18817, 0.5 mg/mL
・"QUANTARED ENHANCED, Pierce 15159CHEMIFLUORESCENT HRP SUBSTRATE"

Biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin- _NH2- AEEA14, biotin-capping _- pSAR, or biotin-NH2-pSAR) were synthesized and captured on neutravidin-coated plates in wash buffer (1x PBS, 0.1% CHAPS) for 2 h at RT with gentle shaking. After washing with wash buffer (4x), the plates were incubated with various amounts of rabbit anti-PEG polyclonal serum (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (1x PBS, 0.1% CHAPS, 0.2% BSA) for 2 h at RT with gentle shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody (diluted 1:5000) was added and the plate was incubated for 1 h at RT with gentle shaking. After washing with wash buffer (5x), HRP substrate was added and the fluorescent signal was measured.

Detailed procedure: Add 100 μL of 1 μM antigen to each well. Agitate at 350 RPM for 2 hours.
Wash with 4 x 200 μL wash buffer.
Prepare each antibody dilution in assay buffer and add 100 μL to each well.
Shake plates at 350 RPM for 2 hours.
Wash with 5 x 200 μL wash buffer. Wait 1 minute after adding wash buffer, then discard.
Add 100 μL of a 1:5000 dilution of goat anti-rabbit IgG (H+L) secondary antibody, HRP (diluted in assay buffer) to each well.
Shake plates at 350 RPM for 1 hour.
Wash with 5 x 200 μL wash buffer. Wait 1 minute after adding wash buffer, then discard.
Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part QuantaRed ADHP Concentrate.
Add 100 μl of QuantaRed Working Solution to each microplate well and incubate for 10 minutes at room temperature.
Peroxidase activity was stopped by adding 10 μl of QuantaRed Stop Solution and shaking the plate for 30 seconds.
The relative fluorescence units (RFU) of each well were measured. The excitation and emission maxima of QuantaRed Substrate are 570 nm and 585 nm, respectively.

Anti-PEG ELISA Assay of Monoclonal Anti-PEG Antibody Binding This assay was used to determine the binding of anti-PEG monoclonal antibodies to various antigens (PEG, AEEA).

reagent:
Pierce Neutravidin High Binding Plate, 8-well Strips, 15508
Assay buffer: 1x DPBS, 2% BSA, 0.1% CHAPS
・Washing buffer: 1x DPBS, 0.1% CHAPS
Anti-PEG IgG (monoclonal), Creative diagnostics CABT-L3140, 190 μg/mL
Anti-PEG IgM (monoclonal), Creative diagnostics CABT-L3141, 240 μg/mL
Goat anti-human IgG Fc secondary antibody, HRP, Thermo A18817, 0.5 mg/mL
Goat anti-human IgM (heavy chain) secondary antibody, HRP, Thermo A18835, 0.5 mg/mL
・"QUANTARED ENHANCED, Pierce 15159CHEMIFLUORESCENT HRP SUBSTRATE"

Procedure: Biotin-labeled antigen (biotin-PEG36K, biotin-capping-AEEA14, or biotin- _NH -AEEA14) was synthesized and captured on neutravidin-coated plates in wash buffer (1x PBS, 0.1% CHAPS) for 2 hours at room temperature with gentle shaking. After washing 4 times with wash buffer, the plates were incubated with various amounts of anti-PEG monoclonal IgG or IgM (25 ng/ml, 250 ng/ml, 500 ng/ml, or 1000 ng/ml) in assay buffer (1x PBS, 0.1% CHAPS, 0.2% BSA) for 2 hours at room temperature with gentle shaking. After washing 5 times with wash buffer, anti-human-HRP secondary antibody (diluted 1:5000) was added, and the plates were incubated for 1 hour at room temperature with gentle shaking. After washing with washing buffer (5x), HRP substrate was added and the fluorescent signal was measured.

Detailed procedure: Add 100 μL of 1 μM antigen to each well. Agitate at 350 RPM for 2 hours.
Wash with 4 x 200 μL wash buffer.
Prepare square dilutions of anti-PEG monoclonal IgG and IgM in assay buffer and add 100 μL to each well.
Shake plates at 350 RPM for 2 hours.
Wash with 5 x 200 μL wash buffer. Wait 1 minute after adding wash buffer, then discard.
Add 100 μL of a 1:5000 dilution of goat anti-human IgG Fc secondary antibody, HRP (diluted in assay buffer) or goat anti-human IgM (heavy chain) secondary antibody, HRP (diluted in assay buffer) to each well.
Shake plates at 350 RPM for 1 hour.
Wash with 5 x 200 μL wash buffer. Wait 1 minute after adding wash buffer, then discard.
Mix 50 parts QuantaRed Enhancer Solution with 50 parts QuantaRed Stable Peroxide and 1 part QuantaRed ADHP Concentrate.
Add 100 μl of QuantaRed Working Solution to each microplate well and incubate for 10 minutes at room temperature.
Peroxidase activity was stopped by adding 10 μl of QuantaRed Stop Solution and shaking the plate for 30 seconds.
The relative fluorescence units (RFU) of each well were measured. The excitation and emission maxima of QuantaRed Substrate are 570 nm and 585 nm, respectively.

Hydrophobicity Comparison: Compounds to be compared are dissolved in acetonitrile and water (2:1 ratio) to a final concentration of 10 μM. To test for relative hydrophobicity, samples are injected into a liquid chromatography-mass spectrophotometer (LC-MS) system consisting of a Thermo Scientific Vanquish LTQ XL linear ion trap. Analysis is performed in positive ion mode using an electrospray ion source. LC separation is performed on a Waters Cortex T3 50 mm x 3 column using a gradient decreasing from 95% A to 100% B in 2 minutes at a flow rate of 0.7 mL/min. Similar retention times indicate that the compounds have similar hydrophobic properties.

Plasma stability assay compounds are tested separately and added to 750 μL of human or mouse plasma at a final concentration of 10 μM. The final mixture is vortexed briefly and incubated at 37°C. At various time intervals (0, 1, 3, 6, 24, 48, and 72 hours), 100 μL of 1% formic acid in acetonitrile is added to the compound-plasma mixture in the microcentrifuge tube. The mixture is vortexed and centrifuged for 10 minutes at 2900 relative centrifugal force (RCF), and 100 μL of the supernatant is transferred to an injection vial. The collected supernatant is analyzed on an LC-MS system consisting of a Shimadzu 8060 NX triple quadrupole mass spectrometer using electrospray in positive ion mode. LC separation is performed on a Waters Cortex T3 50 mm x 3 column using a gradient decreasing from 95% A to 100% B in 2 minutes at a flow rate of 0.8 mL/min. Buffer A is 0.1% formic acid in water and buffer B is 0.1% formic acid.

Particle size analysis is performed by dynamic light scattering using a DynaPro Plate Reader II (Wyatt, Dernbach, Germany). LNP formulations are diluted to 0.005 mg/mL with 1x PBS, and 120 μL of the diluted sample is measured in triplicate in a 96-well plate. From the measurements, the size (Z _-average ) and polydispersity index (PDI) are calculated by cumulant analysis using Dynamics 7.8.1.3 software.

Measurement of Zeta Potential (Electrophoretic Mobility) The zeta potential of the particles is determined by photon correlation spectroscopy using a particle sizer (Zeta Potential/Particle Sizer, Nicomp™ 380 ZLS, Santa Barbara, CA, USA) in a plastic cuvette at a field strength of 4 V/cm and an electrode spacing of 0.4 cm. The LNP formulation is diluted 1:26 with 0.1x PBS for zeta potential measurement. The electrostatic mobility is converted to zeta potential using the Helmholtz-Smoluchowski equation. All measurements are performed at a temperature of 23°C.

mRNA Accessibility Method: RNA accessibility and total RNA concentration in formulations are measured using a modified Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad, CA). LNP samples are diluted to an mRNA concentration of 2-5 ng/μL in 1x TE buffer, pH 7.4. Accessible mRNA is measured by diluting the sample with 1x TE, and total RNA is quantified by diluting the sample with 2% Triton X-100 (VWR International GmbH, Darmstadt, Germany). RiboGreen reagent is added to each sample, and the fluorescent signal is quantified using an Infinite F200PRO microplate reader (Tecan, Maennedorf, Switzerland). For determination of total RNA concentration, a standard curve of RiboGreen fluorescence versus mRNA is used, ranging from 0 to 2.5 μg/mL mRNA in 1% Triton X-100, pH 7.4.

Agarose gel electrophoresis is performed for the evaluation of free RNA. Gels are poured using 1 g agarose dissolved in 100 mL of 1x TAE buffer pH 7.4 (Tris-acetate-EDTA) (Rotiphorese® 50X TAE, Carl Roth, Karlsruhe, Germany), 1 mL of 5% sodium hypochlorite, and 10 pL of GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The gel is allowed to harden for at least 25 minutes at room temperature. The gel is then placed in a gel electrophoresis tank using 1x TAE running buffer (pH 7.4). Prior to loading, samples are incubated at 40°C with or without 2% Triton X-100 for total and free RNA, respectively. The gel is run at 80 V for 40 minutes. Gel images are taken with a Chemidoc XRS imaging system (Bio-Rad, Berkeley, CA, USA).

Hemolysis : A hemolytic assay is performed to test the hemolytic properties of the formulation. For this purpose, human blood is diluted with 1x PBS to a 20% vol/vol blood solution. In a 96-well round-bottom plate, 100 μL of LNP diluted to an equivalent concentration of 0.05 mg mRNA/mL is added to 100 μL of blood solution in PBS and incubated at 37°C for 1 hour (triplicate). After incubation, the plate is centrifuged at 500 x g for 5 minutes at 23°C. The supernatant is then transferred to a clear 96-well plate, and UV absorption is read at 540 nm using a Tecan Pro200 plate reader. Positive and negative controls are made with 0.2% Triton-X and buffer alone, respectively.

Complement activation in vitro: SC5b-9 levels are determined using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Briefly, samples and positive (Cremophor El, Merck, Darmstadt, Germany; cobra venom factor; Quidel Co., San Diego, CA, USA) and negative (1x DPBS and Intralipid) controls are incubated with normal human serum complement (Innovative Research, Michigan, USA) in a 20:80 ratio (sample:serum) for 1 hour at 37°C. LNP formulations are incubated with human serum at a final mRNA concentration of 0.01 mg/ml, the theoretical concentration (corresponding to a dose of 0.49 mg/kg). The SC5b-9 EI kit (Quidel, San Diego, USA) is used.

Cells and cell lines in potency assays HEK 293T-17 cells are seeded in 12-well plates (4 x ¹⁰ cells/well in DMEM (#31966-021/2293715 Gibco) + 10% FCS (S 0615/BS.1640839, Sigma)) 6 hours prior to transfection. Formulations are diluted in Opti-MEM (#51985-026, Gibco) and 0.3 μg/well is used to transfect cells in triplicate. Cells are then centrifuged at 300 x g for 4 minutes. As transfection and/or staining controls, BM LNPs containing PEG-lipids and RNA encoding eGFP (LNP-315-d045, ²⁹ April 2021, BNT Delivery Technologies GmbH, Halle) or the S protein of SARS-CoV-2 (RBP020.LNP, COVVAC/270320, Polymun, Klosterneuburg) were used. Cells were incubated for 18 hours at 37°C/5% _CO2 . For FACS analysis, cells were detached using PBS (#2339094, Gibco, 4°C) and transferred to a 96-well plate. Cells were stained for viability with the fixable viability dye eFluor 450 (#65-0863-14, eBioscience) at a 1:500 dilution, followed by fixation (#42081, Biolegend) and permeabilization (1x dilution, #00-8333-56, eBioscience). To quantify S1 protein expression, cells were stained with a 1:2000 dilution of primary antibody (#40150-R007, Sino Biological) followed by secondary antibody staining (1:1000, #P-2771MP, ThermoFischer). Cells were resuspended in FACS buffer (PBS, EDTA, BSA) for acquisition. To this end, cell viability, transfection frequency, and MFI of the overall and S1-positive populations were determined and compared.

LNP formulation: LNPs were prepared by mixing an aqueous phase (0.15 mg/mL mRNA diluted in 0.1 M citrate buffer, pH 4.0) with an organic phase (a mixture of cationic lipids, DSPC, cholesterol, and stealth components dissolved in ethanol at molar fractions of 47.5:42.5 to 10:10, respectively, with N/P = 6 and a total concentration of 17.05 mM) at a 3:1 volume ratio and 12 mL/min using a microfluidic device (NanoAssemblr® Benchtop, Precision NanoSystems, Vancouver, Canada). The mixture was dialyzed against 1x DPBS (GIBCO, pH 7.4) for 3 hours in a Slide-A-Lyser 10K MWCO dialysis cassette (Thermo Fisher Scientific, Waltham, MA, USA). Residual ethanol was periodically controlled during LNP experiments by osmolality measurement. Physicochemical characterization (size, polydispersity, zeta potential, RNA accessibility, and total RNA concentration) is performed on the day of formulation. After characterization is complete, the formulations are stored at 4° C. for no more than one day. Lipid nanoparticles are diluted with PBS to the desired RNA concentration before in vitro testing.

Example 1 - Ac-(AEEA) ₁₄ - Production of DSPE

Step A: Synthesis of the Ac-(AEEA) ₁₄ -OH Linker. The synthesis of the Ac-(AEEA) ₁₄ -OH linker was carried out on a 0.1 mmol scale using a Liberty Blue HT24 synthesizer. Pre-loaded 2-cltrt resin from the above procedure, "Resin Loading of Peptides onto 2-Cltrt Polystyrene Resin," was added to the Liberty HT suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The synthesis involved multiple iterations on a 0.1 mmol scale for each loading material. The synthesis procedure began with deprotection of the N-terminal α Fmoc protecting group of Fmoc-(AEEA) ₁₄ -OH using 4 mL of 20% piperidine in DMF microwaved at 60 °C for 3 minutes with nitrogen sparging every 3 seconds to mix. After draining, the resin was washed three times with 5 mL DMF for 5 seconds per wash. Then, for the coupling reaction, 2.5 mL of a 0.3 M amino acid solution of Fmoc-AEEA-OH in DMF (7.5 equiv.), 1 mL 1 M DIC (10 equiv.), and 0.5 mL 1 M Oxima + 0.1 M diisopropylethylamine (DIEA) (5 equiv.) was added to the reaction vessel (RV). Single or double coupling steps were performed with microwave heating at 50 °C for 10 min cycles, with nitrogen sparging every 3 seconds for mixing during the coupling cycle. After the coupling time was complete, the resin was drained, and 20% piperidine in DMF was added to the reaction vessel (RV) to initiate deprotection (preferably at 60 °C) for the preparation of the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated sequentially for all amino acids, culminating in a final Fmoc deprotection cycle (which preferably provided a free N-terminus for acetyl capping; a final deprotection of the amino acid sequence with the Fmoc-(AEEA) -OH linker was not performed). After the instrumental synthesis run was completed, the resin was transferred to a ₂₄ ml fritted syringe using DMF. Approximately 10 ml of capping solution containing N-methylpyrrolidone (NMP), 6.25% acetic anhydride, and 12.5% 2 M diisopropylethylamine (DIEA) in 81.25% DMF was added to each syringe. The capping reaction was allowed to proceed for 15 minutes at room temperature (RT). After 15 minutes, the capping solution was drained from the syringes, the resin was rinsed with DMF, and the capping reaction was repeated for another 15 minutes. Upon completion of the two-phase capping reaction, the resin was cleaved by washing five times with 5 ml DMF, followed by five times with 5 ml DCM. A cleavage solution containing 92.5% trifluoroacetic acid (TFA), 3.75% water, and 3.75% triisopropylsilane (TIPS) was prepared. 10 mL of cleavage solution was added to each syringe (for peptides prepared on a 0.1 mmol scale). The peptides were cleaved on a shaker at room temperature for at least 2 hours. The cleaved peptides were then expelled from the syringes. We found it efficient to combine all peptides into one large 250 mL bottle so they could be evaporated simultaneously under nitrogen. Because the pAEE peptide does not precipitate in solutions using ether or hexane, the cleavage solution was evaporated under nitrogen (air or rotary evaporation could potentially be used instead of nitrogen) in a fume hood. After evaporation, the 250 mL bottle contained a gel-like substance of crude peptide material. This was resuspended in enough 50/50 water/acetonitrile + 0.05% TFA so that each repeat could be returned to each conical tube containing approximately 10 mL of resuspension solution.

After cleavage, each synthetic replicate was resuspended in 50/50 water/acetonitrile + 0.05% TFA, frozen, and lyophilized for 1-2 days. After lyophilization, samples were dissolved to approximately 50 mg/mL in 50/50 water/acetonitrile + 0.05% TFA. The solution was vortexed and sonicated until completely dissolved (no visible solid material). An aliquot was taken for crude QC (see the "UPLC-MS Analytical QC Method for Peptide Intermediates" section above). Purification was performed on a Waters Autopurification System equipped with an SQD2 mass detector. Samples were loaded onto a preparative HPLC column (Phenomenex Luna C18(2) LC column, 100 Å pore size, 10 μm particle size) using an autoinjector via a 10 mL sample loop. Sample loading was performed with 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Elution was performed with a 36-minute linear gradient. Column dimensions and flow rates were scale-appropriate (see table below). A Waters SQD2 mass spectrometer was used to automatically trigger fraction collection based on M+1H, M+2H, and M+3H values for the target peptide. UV214 absorbance was monitored but not used for fraction triggering.

Aliquots of selected fractions underwent QC procedures (as described in the "HPLC-MS Analytical QC Procedures for Peptide Intermediates" section above). For some fractions, s-process purity measurements were omitted, as high purity fractions were estimated by visual monitoring of preparative HPLC-MS data. Fractions with measured or estimated purity ≥ 90% were combined into tared polypropylene tubes and lyophilized. If the pure fraction volume exceeded the tube capacity, multiple polypropylene tubes were filled and lyophilized. Each peptide was then resuspended in 50/50 water/acetonitrile + 0.05% TFA, combined into a single tared polypropylene tube, and lyophilized for at least two days. An aliquot was removed from the combined pool and diluted with 50/50 water/acetonitrile + 0.05% TFA for QC.

Step B: Conjugation Ac-(AEEA) ₁₄ -OH (AEEA = (2-(2-(2-aminoethoxy)ethoxy)acetic acid); Ac = acetyl) was dissolved in chloroform at a concentration of approximately 30 mg/mL. To this solution were added 5 molar equivalents each of (7-azabenzotriazol-1-yloxy)trispyrrolizinophosphonium hexafluorophosphate (PyAOP) and 1-hydroxy-7-azabenzotriazole (HOAt), followed by 1.1 equivalents (or 1 equivalent) of DSPE, then 5 equivalents of DIEA. The mixture was stirred at 50°C in a sealed flask for 18-24 hours until UPLC-MS reaction monitoring indicated the consumption of Ac-(AEEA) ₁₄ -OH. After complete conversion, the solvent was removed under reduced pressure and the product was redissolved in methanol for salt exchange and purification by LC-MS using the methods described above for "Salt Exchange and Purification of Amphiphiles" or "Alternative Purification of Amphiphiles." The pure product was lyophilized to give a dry white viscous powder. The UPLC-UV ₂₁₄ purity of the sample was 96% and the MW determined by MS (electrospray) was 2822.55.

Example 2 - Preparation of H-(AEEA) ₁₄ -DSPE
Step A: Boc protection of H-(AEEA) ₁₄ -OH
To a stirring solution of H-(AEEA) ₁₄ -OH in MeCN (approximately 20 mg/mL) was added 2 molar equivalents of Boc ₂ O and 5 equivalents of DIEA. The reaction was stirred at 50°C for 4 hours or until complete conversion was observed by UPLC-MS. The product was crashed with cold (-20°C) diethyl ether, centrifuged, and decanted to give the final product as a pellet. The product was carried on to the next step without further purification. The MW determined by MS (electrospray) was 2150.91.

Step B: Conjugation of DSPE to Boc-(AEEA) ₁₄ -OH and Boc deprotection
Boc-(AEEA) ₁₄ -OH was dissolved in chloroform at a concentration of approximately 30 mg/mL. To this solution, 5 molar equivalents each of PyAOP and HOAt were added, followed by 1.1 equivalents of DSPE, then 5 equivalents of DIEA. The solution was stirred in a sealed flask at 50°C for 18-24 hours until UPLC-MS reaction monitoring indicated the consumption of Ac-(AEEA) ₁₄ -OH. After complete conversion, the solvent was removed under reduced pressure and the product was redissolved in MeOH to a concentration of approximately 30 mg/mL. To this solution, 10 molar equivalents of HCl (4 M in dioxane) were added to remove the Boc protecting group. After 1 hour, deprotection was complete. The reaction solution could be salt-exchanged and purified using the method described above, "Salt Exchange and Purification of Amphiphilic Compounds." The pure product could be lyophilized to obtain a dry white viscous powder. The MW determined by MS (electrospray) was 2780.46.

Example 3 - Preparation of Ac-(AEEA) ₁₄ -α-tocopherol
Ac-(AEEA) ₁₄ -OH was dissolved in chloroform at a concentration of approximately 12 mg/mL. To this solution, 1.3 equivalents of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were added, followed by 0.5 equivalents of 4-dimethylaminopyridine (DMAP), followed by 2.2 equivalents (or 1.2 equivalents) of α-tocopherol. The reaction vial was wrapped in tin foil to minimize light exposure and stirred at room temperature for 4 hours or until the reaction was judged complete by UPLC-MS. Upon completion, volatiles were evaporated under reduced pressure, and the product was redissolved in MeOH for salt exchange and purification. Salt exchange and purification were performed simultaneously using the method described above, "Salt Exchange and Purification of Amphiphiles," eluting with a linear gradient of 52-67% B. The MW determined by MS (electrospray) was 2504.84.

In a separate reaction, Ac-(AEEA) ₁₄ -OH was dissolved in chloroform at a concentration of approximately 12 mg/mL. To this solution were added 2 equivalents of EDC, followed by 1 equivalent of DMAP, and then 2.2 equivalents (or 1.2 equivalents) of α-tocopherol. The reaction vial was wrapped in tin foil to minimize light exposure and stirred at room temperature for 4 hours or until the reaction was judged complete by UPLC-MS. Upon completion, the volatiles were evaporated under reduced pressure, and the product was redissolved in MeOH for salt exchange and purification as described in "Salt Exchange and Purification of Amphiphiles" or "Alternative Purification of Amphiphiles."

In a separate reaction, Ac-(AEEA) ₁₄ -OH was dissolved in dichloromethane at a concentration of 275 mg/mL in the presence of DIC and DMAP. α-Tocopherol was then added to effect the conjugation, and the mixture was stirred overnight (approximately 16 hours) at room temperature, protected from light. Upon completion, the solvent was removed by evaporation, and the resulting crude conjugate was resuspended and purified. After resuspension, the conjugated product was purified by RP-HPLC using an Agilent PLRP-S 100 Å, 50 x 300 mm, 8 μm, 50 x 300 mm system. Elution was performed with eluent A: 1% AcOH in H ₂ O and eluent B: ACN, linearly varying from 30 to 90% B in 120 minutes. This choice of eluent allows for the combination of purification and salt exchange in the same process step. Fractions that exhibited purity ≥95% were combined and lyophilized.

Purity and identity were determined using the following methods.

Example 4 - Preparation of H-(AEEA) ₁₄ -α-Tocopherol Step A: Boc Protection of H-(AEEA) ₁₄ -OH
Boc-(AEEA) ₁₄ -OH was prepared as described in Example 2, Step A.

Step B: Conjugation of α-tocopherol to Boc-(AEEA) ₁₄ -OH and Boc deprotection α-Tocopherol is conjugated to HO-(NMe-AEEA) ₁₄ -Boc (or HO-(AEEA) ₁₄ -Boc) using the method described in Example 3. Boc deprotection is carried out according to the procedure described in Example 2, Step B. Purification and salt exchange are carried out using the method described above in "Salt exchange and purification of amphiphilic compounds."

Example 5 - Preparation of (NMeAEEA) Building Block A 10 g (0.026 M) amount of Fmoc-AEEA-OH monomer was weighed into a 50 ml conical tube and dissolved in 10 ml 1:1 chloroform:TFA. Once all material was dissolved, a small sample was taken for QC analysis by UPLC. Approximately 1.5 equivalents of formaldehyde was added to this mixture, and another small sample was taken for QC analysis by UPLC to assess the intermediate formation of an imine group on the nitrogen atom of Fmoc-AEEA-OH after reaction with formaldehyde. After the addition of formaldehyde, 6 mL of triethylsilane (TES), or approximately 1.46 equivalents, was added to reduce the imine to the desired Fmoc-NMeAEEA-OH monomer (NMeAEEA = (2-(2-(2-methylaminoethoxy)ethoxy)acetic acid)). After the addition of TES, several QC samples of the reaction mixture were run on UPLC to monitor the success of the reduction. The mixture was then diluted with 1:1 acetonitrile:water to a concentration of approximately 250 mg/mL and stored at -80°C until purification was performed.

The crude sample was dissolved to approximately 250 mg/mL in a 1:1:1:1 mixture of TFA:chloroform:acetonitrile:water. The material was sonicated and vortexed until completely dissolved (no visible solid material). The sample was loaded onto a preparative HPLC column (Phenomenex Luna C18(2) LC column, 100 Å pore size, 10 μm particle size, 50 mm × 250 mm) using an autoinjector via a 10 mL sample loop. Sample loading was performed with 95% mobile phase A (0.05% TFA in water), 5% mobile phase B (0.05% TFA in acetonitrile). Peptide elution was performed with an 85-minute linear gradient from 45 to 55% B. The flow rate was 30 mL/min. An SQD2 mass spectrometer was used to trigger automated fraction collection based on the M+1H and M+1H+22 (sodium adduct) values of the target compounds. UV ₂₁₄ absorbance was monitored but not used for fraction triggering. Aliquots of selected fractions were injected into a UPLC-MS using the method described above for "UPLC-MS." For some fractions, in-process QC was eliminated by visual monitoring of preparative HPLC UV ₂₁₄ absorbance and MS to estimate which fractions were highly pure. Fractions with measured or estimated purity ≥ 95% were combined into tared polypropylene tubes and dried on a rotary evaporator. If the pure fraction volume exceeded the tube capacity, multiple polypropylene tubes were filled and dried. Each tube was then resuspended in 50/50 water/acetonitrile + 0.05% TFA, combined into a single tared polypropylene tube, and lyophilized to dryness. Approximately 1 mg of NMeAEEA was dissolved at 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected into the UPLC/MS. The MW of the peptide determined by MS was 399.13 m/z, which agreed with the theoretical MW of 399.17 m/z.

Example 6 - Synthesis of Ac-(NMeAEEA) ₁₄ -α-tocopherol
Step A: Synthesis of the (NMeAEEA) linker
Resin loading of the (NMeAEEA) building blocks was carried out using the method described above, "Resin loading of peptides onto 2-Cltrt polystyrene resin." Synthesis of the Fmoc-(NMeAEEA) ₁₄ -OH, Ac-(NMeAEEA) ₁₄ -OH, and Ac-(NMeAEEA) ₈ -OH linkers was carried out on a 0.1 mmol scale using a Liberty Blue HT24 synthesizer. Preloaded 2-cltrt resin from Resin Loading Method 3 was added to a Liberty HT suspended in 10 ml of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The synthesis included multiple replicates on a 0.1 mmol scale for each loading material. The synthesis procedure began with deprotection of the N-terminal α-Fmoc protecting group using 4 mL of 20% piperidine in DMF by heating at 60 °C for 3 min in a microwave with nitrogen sparging every 3 s for mixing. After draining, the resin was washed three times with 5 mL of DMF for 5 s per wash. For the coupling reaction, 2.5 mL of a 0.3 M amino acid solution of Fmoc-NMeAEEA-OH in DMF (7.5 eq), 1 mL of 1 M DIC (10 eq), and 0.5 mL of 1 M Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 eq) was then added to the reaction vessel (RV). The double coupling step proceeded by microwave heating at 50 °C for two 20 min cycles with nitrogen sparging every 3 s for mixing during the coupling cycle. After the coupling time was complete, the resin was drained, and 20% piperidine in DMF was added to the RV to initiate deprotection of the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated sequentially for all amino acids. Final deprotection of the amino acid sequence was not performed for the Fmoc-(NMeAEEA) ₁₄ -OH linker, while final deprotection of the amino acid sequence was performed for the H(NMeAEEA) ₁₄ -OH linker. After completing the instrumental synthesis run for each compound requiring N-terminal acetylation, the resin was transferred to a 24 ml fritted syringe using DMF. Approximately 10 ml of capping solution containing N-methylpyrrolidone (NMP), 6.25% acetic anhydride, and 12.5% 2 M diisopropylethylamine (DIEA) in 81.25% DMF was added to each syringe. The capping reaction proceeded for 15 min at room temperature (RT). After 15 minutes, the capping solution was expelled from the syringe, the resin was rinsed with DMF, and the capping reaction was repeated for another 15 minutes. Once the second capping reaction was complete, the resin was washed five times with 5 ml DMF, then five times with 5 ml DCM to cleave the resin.

Step C: Conjugation of Ac-(NMe-AEEA) ₁₄ -OH to α-Tocopherol α-Tocopherol is conjugated to Ac-(NMe-AEEA) ₁₄ -OH using the method described above in Example 3.

Example 7 - Preparation of H-(NMeAEEA) ₁₄ -α-Tocopherol α-Tocopherol is conjugated to Boc-(NMeAEEA) ₁₄ -OH using the method described above in Example 3. Boc deprotection is carried out using the method described in Example 2, Step B. Salt exchange and purification was carried out using the method described above in "Salt exchange and purification of amphiphilic compounds."

Example 8 - Preparation of Ac-(AEEA) ₁₄ -DMG
Conjugation of DMG to Ac-(AEEA) ₁₄ -OH is carried out according to the protocol described in Example 3. Upon completion, the solvent is removed under reduced pressure and the product is redissolved in MeOH for salt exchange and purification using the method described above under "Salt Exchange and Purification of Amphiphilic Compounds." Alternatively, the product is purified using the methods described above under the headings "Alternative Purification of Amphiphilic Compounds" or "Alternative Purification of Further Amphiphilic Compounds" (e.g., redissolving the product in chloroform and purifying the redissolved product by size exclusion).

Example 9 - Preparation of Ac-(NMeAEEA) ₁₄ -DSPE
DSPE is conjugated to Ac-(NMeAEEA) ₁₄ -OH using the method described in Example 1. Salt exchange and purification is performed using the method described above "Salt exchange and purification of amphiphiles."

Example 10 - Preparation of Additional Amphiphilic OEG Conjugate Compounds The following amphiphilic OEG conjugate compounds are synthesized using the methods shown in the following table.
Ceramide: N-palmitoyl-ceramide

For example, Ac-(AEEA) ₈ -α-tocopherol was prepared according to the following scheme.
Specifically, Ac-(AEEA) ₈ -OH was dissolved in chloroform at a concentration of approximately 12 mg/mL. To this solution were added 2 equivalents of EDC, followed by 1 equivalent of DMAP, followed by 2.2 equivalents (or 1.2 equivalents) of α-tocopherol. The reaction vial was wrapped in tin foil to minimize light exposure and stirred at room temperature for 4 hours or until the reaction was judged complete by UPLC-MS. Upon completion, the volatiles were evaporated under reduced pressure and the product was redissolved in MeOH. Purification was carried out as described in "Salt Exchange and Purification of Amphiphiles" or "Alternative Purification of Amphiphiles."

Ac-(AEEA) ₈ -DMG was prepared according to the following scheme.
Conjugation of DMG to Ac-(AEEA) ₈ -OH was carried out according to the synthesis of Ac-(AEEA) ₈ -α-tocopherol, except that the reaction vial was not wrapped in aluminum foil (not light sensitive). Purification was carried out as described in "Salt exchange and purification of amphiphiles" or "Alternative purification of amphiphiles."

Ac-(AEEA) ₁₄ -DMA was prepared according to the following scheme.
Ac-(AEEA) ₁₄ -OH was dissolved in chloroform at a concentration of approximately 30 mg/mL. To this solution, 5 molar equivalents each of PyAOP and HOAt were added, followed by 1.2 equivalents of DMA and then 5 equivalents of DIEA. The reaction mixture was stirred at 50°C in a sealed flask for 18-24 hours until UPLC-MS reaction monitoring indicated the consumption of all Ac-(AEEA) ₁₄ -OH. Upon completion, purification was carried out as described in "Salt Exchange and Purification of Amphiphiles" or "Alternative Purification of Amphiphiles."

Further amphiphilic OEG conjugate compounds (e.g., those where n is 10) can be synthesized using the above method.

Example 11 - Preparation of control lipid DSPE-PEG2K-cycloALFA- _NH2
Step A: Synthesis of the lactam ALF peptide sequence: MPA-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu-NH _2. Synthesis of the ALF peptide sequence was carried out on a 0.1 mmol scale using a Liberty Blue HT24 synthesizer. Sieber amide resin was loaded onto the Liberty HT suspended in 10 mL of 1:1 dichloromethane/dimethylformamide (DCM:DMF) for pre-swelling and resin transfer. The synthesis procedure began with deprotection of the N-terminal α-Fmoc protecting group using 4 mL of 20% piperidine in DMF, microwaved at 60 °C for 3 min with nitrogen sparging every 3 s for mixing. After draining, the resin was washed three times with 5 mL of DMF for 5 s per wash. Next, 2.5 mL of the next 0.3 M amino acid solution (7.5 equiv.), 1 mL 1 M DIC (10 equiv.), and 0.5 mL 1 M Oxyma + 0.1 M diisopropylethylamine (DIEA) (5 equiv.) were added to the reaction vessel (RV) for the coupling reaction. The double coupling step proceeded by heating in a microwave with nitrogen sparging every 3 seconds for mixing during both coupling cycles, first at 50 °C for a 6-minute cycle and then at 50 °C for a further 10-minute cycle. After the second coupling period was complete, the resin was drained, and 20% piperidine in DMF was added to the RV to initiate deprotection of the next incoming amino acid. This cycle of Fmoc removal and coupling was repeated sequentially for all amino acids. Liberty Blue exterior position 6 was used to add Fmoc-Glu(O-2-PhiPr)-OH to the peptide sequence at the corresponding cyclization point for lactam formation, and position 7 was used for the addition of Fmoc-Lys(Mmt)-OH amino acids. Peptides containing an N-terminal Mpa(Trt)-OH did not require final deprotection of the peptide due to the absence of Fmoc protection of the Mpa(Trt)-OH amino acid. The peptide resin was washed four times with 4 mL DMF and then returned from the RV to the starting HT position to begin synthesis of the next peptide in the series. After the instrument synthesis run was completed for each peptide, the resin was placed in a 24 mL fritted syringe using DCM and rinsed three times with DCM to remove excess DMF.

After washing, the resin was treated six times with 5 ml of 2% trifluoroacetic acid (TFA), 1% triisopropylsilane (TIS) in DCM, approximately 5 ml per reaction. The flow-through solution from each reaction, containing the semi-protected peptide, was collected in a separate 50 ml conical tube for each peptide. The semi-protected peptide solution was then evaporated under a gentle nitrogen (N ₂ ) blanket until the total volume remaining in each tube was less than 5 ml. Once the volume in each conical tube was less than 5 ml, the peptide was resuspended in 20 ml of 1:1 acetonitrile:t-butanol (AcN:tBu-OH) and sonicated until the solution was homogenous. The peptide tubes were then frozen to -80°C and placed in a lyophilizer for drying.

Once the semi-protected peptide was dry, approximately 5 equivalents of PyAOP and HoAt (per 0.1 mmol scale synthesis) were weighed out and dissolved in 4 ml of DMF. Once the reagents were completely dissolved, this solution was added to each of the peptide tubes. Once the semi-protected peptide material was dissolved, approximately 10 equivalents (per 0.1 mmol scale synthesis) of DIEA was added to each solution, and a slight color change was observed, with the solutions turning yellow. The solutions were then allowed to react at room temperature for 2-3 hours.

After 2-3 hours of reaction time at RT, the protected peptide was precipitated by adding approximately 45 ml of chilled HPLC water to each 50 ml conical tube, and then the tubes were centrifuged at 1000-1200 rpm for approximately 5 minutes. This was repeated twice to remove any excess DMF; after the second precipitation, the peptide pellet was resuspended in 1:1 (AcN:tBu-OH), frozen at -80°C, and lyophilized overnight.

After the peptides were completely dried by lyophilization, approximately 10 ml of cleavage cocktail containing 92.5% TFA, 2.5% water (H _O ), 2.5% TIS, and 2.5% thioanisole was added to each 50 ml conical tube and shaken at room temperature for 2–3 hours. The 10 ml cleavage cocktail was divided into two 50 ml conical tubes, each containing 5 ml. Approximately 45 ml of cold (−20°C) 1:1 hexane:diethyl ether was added to each tube. The mixture was then centrifuged at 3500 rpm for 10 minutes. After decanting the ether, an additional 40 ml of cold 1:1 hexane:diethyl ether was added to further wash the peptide pellet. The vessel was vortexed to break up the peptide pellet and then centrifuged again at 3500 rpm for 10 minutes. After decanting the ether a second time, the tubes were placed on their side in a fume hood to dry. The peptide pellet was then reconstituted in approximately 10 ml of 1:1 acetonitrile/water (AcN:H ₂ O), frozen at -80°C, and lyophilized. After the peptide was completely dried, a small sample of the dried crude peptide powder was placed in a 1.5 mL Eppendorf tube and then dissolved with enough 3:1 dimethyl sulfoxide/water (DMSO:H ₂ O) to achieve a peptide sample concentration of approximately 2 mg/mL. 50 μL of the peptide sample was then placed in a plastic vial and loaded onto a Waters Acuity UPLC-MS for crude analysis. Once the molecular weight was confirmed and the column retention time was obtained, the crude peptide was purified in preparation for conjugation to DSPE-PEG2K-Mal imide.

The crude peptide was subjected to UPLC-MS as described above. Approximately 1 mg of the UPLC-purified peptide was dissolved at 1 mg/mL in 50/50 water/acetonitrile + 0.05% TFA and injected into the UPLC/MS for purity and MW. The MW (electrospray) of the peptide determined by MS was 1728.2, consistent with the theoretical MW of 1728.0.

Step B: Conjugation of MPA-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- _NH2 to DSPE-PEG2K. Upon completion of purification of the cyclic ALF peptide, the final weight of the purified material was used to calculate the millimolar amount of cyclic alpha required for conjugation to the DSPE-PEG2K-Mal compound. Once the amount of cyclic ALF peptide was estimated, 1.2 equivalents of DSPE-PEG2K-Mal relative to the amount of cyclic alpha was weighed into a 15 ml conical tube and dissolved in DMSO to a concentration of approximately 4-5 mg/ml. A small sample of the dissolved DSPE-PEG2K-Mal was taken for QC analysis by UPLC to assess the initial retention time. Once the DSPE-PEG2K-Mal material was dissolved, this solution was then used to dissolve the cyclic ALF peptide, resulting in a cyclic ALF peptide concentration of approximately 5-8 mg/ml in the mixture. A small sample of this mixture was taken for QC analysis by UPLC to assess the retention times of both components in the mixture. Once all material was completely dissolved, a reaction mixture pH adjustment was performed using Trizma base solution prepared in water at a concentration of 2.5 mg/ml and added to the reaction mixture to dilute the total reaction mixture volume by approximately 30-40%. Small samples of the reaction mixture were taken for QC analysis at 15- and 30-minute intervals to assess reaction progress. Once it was confirmed that all cyclic alpha starting material had been removed from the reaction, the mixture was subjected to purification and isolation of the conjugated material.

The title compound (control lipid DSPE-PEG2K-cycloALFA- _NH ) was loaded onto a preparative HPLC column (Waters XSelect Peptide CSH C18 OBD Prep column, 130 Å, 5 μm, 19 mm × 100 mm) using an autoinjector via a 10 mL sample loop. Sample loading was performed with 85% mobile phase A (0.05% TFA in water) and 15% mobile phase B (0.05% TFA in acetonitrile). Peptide elution was a 19-minute linear gradient from 75 to 95% B. The flow rate was 15 mL/min. Fraction collection was automatically triggered using an SQD2 mass spectrometer. For the first purification run, the trigger mass was selected using the most intense MS peak seen in the crude reaction UPLC-MS trace. After one round of purification, the mass was adjusted to the most intense mass across the collection range of the conjugate for the preparative system. UV ₂₁₄ absorbance was monitored but not used for fraction induction. Fractions measured or estimated to be ≥ 90% pure were combined into tared polypropylene tubes and lyophilized.

For salt exchange and purification, the lyophilized title compound was subjected to the above-described method, "Salt exchange and purification of amphiphilic compounds."

The purity and identity of the purified and lyophilized title compound were determined on a Waters H-Class UPLC/MS system using a linear gradient of 10 to 80% B in 8 minutes at a flow rate of 0.5 mL/min, where mobile phase A was 0.100% TFA in water and mobile phase B was 0.085% TFA in acetonitrile. The column (ACQUITY UPLC PROTEIN BEH C4 column, 300 Å, 1.7 μm, 2.1 mm × 100 mm) was heated to 60°C. The UPLC-UV ₂₁₄ purity of the sample was 98%, and the MW determined by MS (electrospray) was 4399.2. Due to the polydisperse nature of PEG2K, the MW was based on the most abundant of the many ions.

The title compound (also referred to herein as DSPE-PEG2K-cycloALFA-NH ₂ (DSPE-PEG2K-succ-MPA-cycloALFA-NH ₂ )) has the following formula:

Example 12 - Preparation of the control lipid PEG2K-DSPE-Pro-cycloALFA- _NH2 The title compound was synthesized essentially according to the procedure described in Example 11.

Step C: Resin loading of Fmoc-AEEA-OH onto 2-Cltrt polystyrene resin Resin loading was carried out using the method described above "Resin loading of peptide onto 2-Cltrt polystyrene resin" and Fmoc-AEEA-OH.

Step D: Synthesis of Ac-(AEEA) ₈ -OH Linker The synthesis of Ac-(AEEA) ₈ -OH linker was according to Example 1, Step A.
The title compound (PEG2K-DSPE-Pro-cycloALFA-NH ₂ (also referred to herein as DSPE-PEG2K-succ-MPA-Pro-cycloALFA-NH ₂ ) has the following formula:

Example 13 - Preparation of the conjugate H ₂ N-(rvALFA-Pro-MPA)-succ-(AEEA) ₁₄ -DSPE
Step A: Conjugation of H-(AEEA) ₁₄ -OH to 3-maleimidopropionic acid N-hydroxysuccinimide ester
H-(AEEA) ₁₄ -OH is dissolved in DMF at a concentration of approximately 45 mg/mL. To this solution, 1.3 equivalents of 3-maleimidopropionic acid N-hydroxysuccinimide ester and 4 equivalents of DIEA are added. The solution is stirred at room temperature for 2-4 hours until the reaction is complete as monitored by UPLC-MS. Upon completion, the reaction solution is precipitated with diethyl ether in a conical tube, centrifuged, and the supernatant is decanted to obtain the product as a viscous pellet. The product is then used in the next step without further purification. The MW determined by MS (electrospray) is 2201.73.

Step B: Conjugation of DSPE to maleimide-(AEEA) ₁₄ -OH
Conjugation of DSPE to maleimide-(AEEA) ₁₄ -OH was carried out using the procedure described in Example 1. Volatiles were removed under reduced pressure and the product was used for conjugation to alpha without purification. No yield was observed for this step. MW determined by MS (electrospray) was 2930.38.

Step C: Conjugation of Maleimido-(AEEA) ₁₄ -DSPE to MPA-Pro-ALFA
Maleimide-(AEEA) ₁₄ -DSPE was dissolved in DMSO at approximately 5 mg/mL and stirred in a round-bottom flask at room temperature. To this solution was added MPA-Pro-ALFA (e.g., 1.1 molar equivalents). An aqueous solution of Trizma base was prepared until the pH was approximately 8.5 (approximately 2.5 mg/mL). This solution was added to the reaction flask until 5.5 molar equivalents of Trizma base had reacted. The reaction solution was stirred for 4 hours or until UPLC-MS indicated the consumption of all starting material. The product solution was injected into HPLC for salt exchange and purification using the method described above, "Salt Exchange and Purification of Amphiphiles." The pure product was lyophilized to yield a dry white viscous powder. The UPLC-UV ₂₁₄ purity of the sample was 95%, and the MW, as determined by MS (electrospray), was 4802.75.

The title compound (conjugate H ₂ N-(rvALFA-Pro-MPA)-succ-(AEEA) ₁₄ -DSPE) has the following formula:

Example 14 - Determination of Binding of Anti-PEG Antibodies to Various Antigens (PEG, AEEA, pSAR) Using Anti-PEG ELISA Assay The "anti-PEG ELISA assay" described above (see also Figure 1) was used to determine the binding of anti-PEG polyclonal antibodies to various antigens (PEG, AEEA, pSAR). Briefly, biotin-labeled antigens (biotin-PEG36K, biotin-capping-AEEA14, biotin- _NH2- AEEA14, biotin-capping-pSAR, or biotin- _NH2 -pSAR, where pSAR is a compound of Formula XVIII disclosed herein, where _R21 is H or acetyl (Ac); and R ₂₂ is Lys-biotin) was synthesized and captured on a neutravidin-coated plate in wash buffer (1x PBS, 0.1% CHAPS) for 2 hours at room temperature with slow shaking. After washing with wash buffer (4x), the plate was incubated with various amounts of rabbit anti-PEG polyclonal serum (7 ng/ml, 3 ng/ml, 167 ng/ml, or 830 ng/ml) in assay buffer (1x PBS, 0.1% CHAPS, 0.2% BSA) for 2 hours at room temperature with slow shaking. After washing with wash buffer (5x), anti-rabbit-HRP secondary antibody was added (1:5000 dilution), and the plate was incubated for 1 hour at room temperature with slow shaking. After washing with wash buffer (5x), HRP substrate was added, and the fluorescent signal was measured. The results are shown in Figure 2.

As can be seen in Figure 2, rabbit polyclonal anti-PEG antibodies bind to PEG36k but not to polymers containing the structure of formula (I) or to a negative control (biotin-labeled polysarcosine structure). Thus, without wishing to be bound by any theory, it is believed that the specific structure of formula (I) (i.e., OEG stretches separated from each other by the moiety ^-X2 - ^X1 -Y-) prevents anti-PEG antibodies from binding to this structure and similarly to the amphiphilic OEG conjugate compounds of the present invention.

The anti-PEG ELISA assay was repeated using anti-PEG IgG and anti-PEG IgM antibodies, respectively. The results of these experiments are shown in Figure 3.

The data shown in Figure 3 indicate that neither anti-PEG IgG nor anti-PEG IgM antibodies bind to polymers containing the structure of formula (I) at the antibody concentrations tested, while both types of antibodies bind to PEG36k. Thus, Figure 3 confirms the results of Figure 2.

Example 15 - Comparison of the properties (length, molecular weight, and hydrophobicity) of PEG structures and structures of formula (I). Structures (AEEA) _14-17 have lengths similar to that of PEG2k. Furthermore, structure (AEEA) ₁₄ can be synthesized in a monodisperse form (MW: 2050 Da), while PEG2k can only be synthesized in a polydisperse form (average MW: approximately 2000 Da).

To determine whether the structure of formula (I) differs from the PEG structure in terms of hydrophobicity, H-(AEEA) ₁₄ -OH and Fmoc-(PEG) ₃₆ were subjected to the above-described "Hydrophobicity Comparison" method. The retention times of compounds H-(AEEA) ₁₄ -OH and Fmoc-(PEG) ₃₆ determined by this method were 1.03 minutes and 1.18 minutes, respectively. Since similar retention times for different compounds indicate that these compounds have similar hydrophobic properties, it can be concluded that the structure of formula (I) exhibits similar hydrophobic properties to the PEG structure.

Example 16 - Determination of Plasma Stability of Structures of Formula (I) The plasma stability of Ac-(AEEA) ₁₄ -OH and H-(AEEA) ₁₄ -OH was tested using the "Plasma Stability Assay" described above. The results of these experiments are shown in Figure 4.
As can be seen in Figure 4, both compounds are stable in human and mouse plasma for at least 72 hours.

Example 17 - Preparation of Lipid Nanoparticles Using HY501 and Various Fractions of DSPE-AEEA14-AC as the Cationic Ionizable Lipid The purpose of this example was to test DSPE-AEEA14-AC (i.e., the amphiphilic OEG conjugate compound of formula (V-1) of the present invention) as a stealth moiety for mRNA nanoparticle engineering using LNP technology containing HY501 (i.e., the cationically ionizable lipid of formula XIV-3) as the cationic ionizable lipid. DSPE-AEEA14-AC-containing LNPs were prepared by mixing the aqueous RNA phase and the organic lipid phase using a microfluidic device. Various molar fractions of DSPE-AEEA14-AC lipid (1-5%) were used in the LNP formulation. The ionizable lipid HY501, helper lipids DSPC, and cholesterol (where X is the fraction of DSPE-AEEA14-AC) were used in molar fractions ranging from 47.5:42.5 to X:10. An N/P ratio of 6 was selected for this experiment, and the RNA construct for all LNP formulations was V09 DS(P020.2) (a modified mRNA encoding the P2 S protein of SARS-CoV-2). For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these DSPE-AEEA14-AC-containing formulations were compared with two PEG-containing formulations (BM_1 and BM_2). A summary of all formulations used in this study is shown in Table 2.

Figure 5 shows the size evolution and size distribution of DSPE-AEEA14-AC-containing LNPs (HY-501-based) prepared with different DSPE-AEEA14-AC concentrations in the formulation. As can be seen, the particle size decreased sharply from 300 to 101 nm as the amount of DSPE-AEEA14-AC in the LNP composition increased from 1 to 2 mol%. Furthermore, the particle size decreased moderately from 101 to 64 nm as the amount of DSPE-AEEA14-AC lipid increased from 2 to 5 mol%. Meanwhile, all formulations exhibited a PDI greater than 0.30.

Figure 6 shows the zeta potential of all DSPE-AEEA14-AC-containing LNPs in this example measured in 0.1x PBS at pH 7. Results for the BM formulation are also included. As can be seen, a slight negative surface charge was obtained for all formulations.

The RNA accessibility and amount of free RNA in LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 7 shows the RNA accessibility of the formulations measured using the RiboGreen assay method (A) or the agarose gel electrophoresis method (B). As can be seen, the RNA inaccessibility in the formulations increased with the DSPE-AEEA14-AC concentration in the formulations. On the other hand, the agarose gel electrophoresis images show that there was almost no or negligible free RNA in the formulations (free RNA <1% in each formulation). These results from the agarose gel measurements indicate that virtually all RNA is bound to the particles in the DSPE-AEEA14-AC-containing formulations.

The complement activation of the formulations was tested by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Figure 8 shows terminal complement complex (SC5b-9) formation after incubation of LNP formulations with human serum. The concentration of SC5b-9 complex formed relative to the control is also included. As can be seen, no or negligible complement activation was observed with all formulations.

Figure 9 shows the hemolysis analysis after incubation (neutral pH conditions) of LNP formulations with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, negligible hemolysis was observed for all test formulations.

Figure 10 shows cell viability tested by FACS (fluorescence-activated cell sorting) analysis after incubation with LNP formulations. As can be seen, good viability (>90%) was observed for all tested formulations.

Example 18 - Preparation of Lipid Nanoparticles Using HY501 and Various Fractions of VE-AEEA14-AC as the Cationically Ionizable Lipid The purpose of this example was to test VE-AEEA14-AC (i.e., the amphiphilic OEG conjugate compound of formula (V-5) of the present invention) as a stealth moiety for mRNA nanoparticle engineering using LNP technology involving HY501 as the cationically ionizable lipid. VE-AEEA14-AC-containing LNPs were prepared by mixing the aqueous RNA phase and the organic lipid phase using a microfluidic device. Various molar fractions of VE-AEEA14-AC lipid (2-6%) were used in the LNP formulation. Molar fractions ranging from 47.5:42.5 to X:10 of the cationically ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of VE-AEEA14-AC) were used. An N/P ratio of 6 was selected for this experiment, and the RNA construct for all LNP formulations was V09 DS (P020.2) (a modified mRNA encoding the P2 S protein of SARS-CoV-2). For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these VE-AEEA14-AC-containing formulations were compared with other PEG-containing formulations (BM_1 and BM_2). A summary of all formulations used in this study is shown in Table 3.

Figure 11 shows the size evolution and size distribution of VE-AEEA14-AC-containing LNPs (based on HY-501) prepared with different VE-AEEA14-AC concentrations in the formulation. As can be seen, small particles (d < 100 nm) were obtained using 2-6 mol% VE-AEEA14-AC lipid in the formulation. Furthermore, all formulations exhibited a PDI better than 0.32.

Figure 12 shows the zeta potential of all VE-AEEA14-AC-containing LNPs from this example measured in 0.1x PBS at pH 7. Results for the BM formulation are also included. As can be seen, a neutral to slightly negative surface charge was obtained for all formulations.

The RNA accessibility and amount of free RNA in the LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 13 shows the RNA accessibility of the formulations measured using the RiboGreen assay method (A) or the agarose gel electrophoresis method (B). As can be seen, high levels of inaccessible RNA were observed in all formulations. On the other hand, the agarose gel electrophoresis images show that free RNA was nearly absent or negligible in the formulations (free RNA in each formulation was <1%, except for the BM_1 formulation, which had free RNA <5%). These results from the agarose gel measurements indicate that virtually all RNA is bound to the particles in the VE-AEEA14-AC-containing formulations.

Complement activation of the formulations was tested by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). No complement activation was observed in any of the formulations.

Hemolysis analysis was determined after incubation of LNP formulations with human whole blood (neutral pH conditions). Negligible hemolysis was observed for all test formulations.

Figure 14 shows S1 protein expression in HEK 293T-17 cells after incubation with LNP formulations. Mean fluorescence intensity (MFI) (see Figure 14A) and cell viability (see Figure 14B) were examined by FACS (fluorescence-activated cell sorting) analysis. As can be seen, expression was observed in all LNP formulations tested. Furthermore, clear VE-AEEA14-AC-mol%-dependent activity was observed with LNP transfection, while maintaining good viability (>90%). As can be seen, for VE-AEEA14-AC-containing formulations, repressed expression was observed in formulations containing VE-AEEA14-AC, and expression decreased with increasing VE-AEEA14-AC concentration by 2 mol%.

Example 19 - Yield and Purity of Peptide Intermediates and Amphiphiles. The peptide intermediates Ac-(AEEA) ₈ -OH and Ac-(AEEA) ₁₄ -OH were synthesized as described above, and samples of these intermediates for UPLC and mass spectrometry were taken directly after cleavage of each intermediate from the resin, either before (crude purity) or after (final purity) the QC method. Figures 15A-D show the results for Ac-(AEEA) ₈ -OH, while Figures 15E-H show the results for Ac-(AEEA) ₁₄ -OH. Figures 15A, B, E, and F show the respective UPLC chromatograms, and Figures 15C, D, G, and H show the respective mass spectra recorded using either samples not subjected to the QC method (Figures 15A, C, E, G) or samples subjected to the QC method (Figures 15B, D, F, H).

From these data, the yield and purity (crude purity (i.e., before the QC method) and final purity (i.e., after the QC method)) were calculated and are shown in Table 4.

Additionally, several amphiphilic compounds were synthesized as described above. During synthesis and after purification, samples were taken and analyzed using the methods described in "Salt Exchange and Purification of Amphiphilic Compounds" or "Another Method for Purification of Amphiphilic Compounds." Figure 16 shows UPLC chromatograms of Ac-(AEEA) ₈ -α-tocopherol synthesis samples taken after (A) 5 minutes and (B) 4 hours (i.e., at the end) of reaction. As can be seen, the starting material Ac-(AEEA) ₈ -OH was present at 5 minutes of reaction time but was not detected at the end of the reaction. FIG. 17 shows exemplary UPLC chromatograms (A, C, E, G, I) and mass spectra (B, D, F, H, J) of the amphiphilic compounds Ac-(AEEA) ₈ -α-tocopherol (A, B), Ac-(AEEA) _{14 -α} -tocopherol (C, D), Ac-(AEEA) 14 -DMA (E, F), Ac-(AEEA) ₈ -DMG (G, H), and Ac-(AEEA) ₁₄ -DSPE (I, J).

From these data, the yield and purity (crude purity (i.e., before the QC method) and final purity (i.e., after the QC method)) were calculated and are shown in Table 5. The total purity was calculated based on UV absorbance at 214 nm.
*60% yield of Ac-(AEEA) ₈ -α-tocopherol is attributed to size exclusion chromatography (SEC) purification. All others were purified by RP-HPLC.

Crude purity is low for reactions with lipid starting materials that absorb strongly at 214 nm and for some reactions using PyAOP/HOAt/DIEA, as some of these reagents and by-products can be observed by UPLC.

Example 20 - Investigating the Effects of Various Amphiphilic OEG Conjugate Compounds on the Engineering and Biological Behavior of LNP Formulations. The purpose of this example was to test various amphiphilic OEG conjugate compounds as stealth moieties for the engineering of mRNA nanoparticles using LNP technology containing HY501 as the cationically ionizable lipid. LNPs containing amphiphilic OEG conjugate compounds were fabricated by mixing the aqueous RNA phase and the organic lipid phase using a microfluidic device. Various molar fractions of amphiphilic OEG conjugate compounds were used in the LNP formulations. Molar fractions ranging from 47.5:42.5 to X:10 of the cationically ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of each amphiphilic OEG conjugate compound) were used. V09 mRNA (a modified mRNA encoding the P2 S protein of SARS-CoV-2) was used in this example. An N/P ratio of 6 was selected for this experiment. Furthermore, the physicochemical properties and biological performance of these formulations containing amphiphilic OEG conjugate compounds were compared with two PEG-containing formulations (BM_1 and BM_2). A summary of all formulations used in this example is shown in Table 6.

The size evolution and size distribution of amphiphilic OEG conjugate compound-containing LNPs (based on HY-501) prepared with various types and concentrations of amphiphilic OEG conjugate compounds in the formulations are shown in Figure 18. The size and size distribution of the BM formulations are also included. As can be seen, the size of the amphiphilic OEG conjugate compound-containing LNP formulations decreased with increasing stealth moiety concentration. Furthermore, small particles (d<100 nm) were obtained in most formulations. Meanwhile, all formulations showed a PDI better than 0.25.

Figure 19 shows the zeta potential of all amphiphilic OEG conjugate compound-containing LNPs from this example, measured in 0.1x PBS at pH 7. Results for the BM formulation are also included. As can be seen, a neutral to slightly negative surface charge was obtained for all formulations. The slightly negative surface charge of the amphiphilic OEG conjugate compound-containing formulations is likely due to the acetylated functional groups on these molecules.

The RNA accessibility and amount of free RNA in the LNP formulations were measured by the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 20 shows the RNA accessibility of the formulations measured by the RiboGreen assay method (A) or the agarose gel electrophoresis method (B). As can be seen, regardless of the type of amphipathic OEG conjugate compound, the level of inaccessible RNA increased as the concentration of the amphipathic OEG conjugate compound in the formulation increased. Furthermore, inaccessible RNA was observed in almost all formulations. On the other hand, the agarose gel electrophoresis images show that there was little to no free RNA in the formulations. These results from the agarose gel measurements indicate that virtually all RNA is bound to the particles of the amphipathic OEG conjugate compound-containing LNP formulations.

The formulations were tested for complement activation by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). As can be seen in Figure 21, no complement activation was observed with any of the formulations.

Figure 22 shows the hemolysis analysis after incubation (neutral pH conditions) of the LNP formulations with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, negligible hemolysis was observed with all other formulations.

Figure 23 shows S1 protein expression in HEK 293T-17 cells after incubation with LNP formulations. Mean fluorescence intensity (MFI) (see Figure 23A) and cell viability (see Figure 23B) were examined by FACS (fluorescence-activated cell sorting) analysis. As can be seen, expression was observed in all tested LNP formulations. Furthermore, clear amphiphilic OEG conjugate compound-mol%-dependent activity was observed with LNP transfection, while maintaining good viability (>90%). For VE-AEEA14-AC-containing formulations, low expression was observed in formulations containing VE-AEEA14-AC, and expression decreased with increasing VE-AEEA14-AC concentration by 1 mol%. Furthermore, for VE-(AEEA)8_AC-containing formulations, the highest level of expression was observed in HY501-VE-(AEEA)8-AC_3. Comparison of VE-(AEEA) ₁₄ -AC and VE-(AEEA)8_AC-containing formulations indicates that formulations with a short stealth moiety exhibit superior performance. Furthermore, for DMG-(AEEA)8_AC-containing formulations, the highest level of expression was observed for HY501-DMG-(AEEA)8-AC_4. The lower activity level of HY501-DMG-(AEEA)8-AC_3 is likely due to the large particle size of this formulation. Comparison of all tested LNP formulations containing amphiphilic OEG conjugate compounds indicates that the hydrophobic tail type may also play a role in the activity of the formulation, with the following trend observed for expression efficiency: VE > DMG > DMA.

Example 21 - Investigating the Effects of Various Amphiphilic OEG Conjugate Compounds on the Engineering and Biological Behavior of LNP Formulations. Luciferase Formulation. The purpose of this example was to test various amphiphilic OEG conjugate compounds as stealth moieties for engineering mRNA nanoparticles using LNP technology, including HY501 as the cationically ionizable lipid. LNPs containing amphiphilic OEG conjugate compounds were fabricated by mixing the aqueous RNA phase and the organic lipid phase using a microfluidic device. Various molar fractions of amphiphilic OEG conjugate compounds were used in the LNP formulation. Molar fractions ranging from 47.5:42.5 to X:10 of the cationically ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of each amphiphilic OEG conjugate compound) were used. Luciferase mRNA (modified mRNA encoding firefly luciferase) was used in this example. An N/P ratio of 6 was selected for this experiment. Furthermore, the physicochemical properties and biological performance of these formulations containing amphiphilic OEG conjugate compounds were evaluated. A summary of all formulations used in this example is provided in Table 7.

The size evolution and size distribution of amphiphilic OEG conjugate compound-containing LNPs are shown in Figure 24. As can be seen, the size of amphiphilic OEG conjugate compound-containing LNP formulations decreased with increasing stealth moiety concentration. Furthermore, small particles (<100 nm) were obtained in all formulations except for the HY501-DMG-(AEEA)8-AC_3 formulation. Furthermore, the majority of formulations exhibited a PdI of better than 0.3.

Figure 25 shows the zeta potential of all amphiphilic OEG conjugate compound-containing LNPs measured in 0.1x PBS at pH 7. As can be seen, a neutral to slightly negative surface charge was obtained for all formulations. The slightly negative surface charge of the amphiphilic OEG conjugate compound-containing formulations is likely due to the acetylated functional groups of these molecules.

The RNA accessibility and amount of free RNA in the LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 26 shows the RNA accessibility of the formulations measured using the RiboGreen assay method (A) or the agarose gel electrophoresis method (B). As can be seen, inaccessible RNA was found in almost all formulations. On the other hand, the agarose gel electrophoresis image shows that there is little to no free RNA in the formulations. These results from the agarose gel measurements indicate that virtually all RNA is bound to the particles of the LNP formulation containing the amphiphilic OEG conjugate compound.

To test the effect of various amphiphilic OEG-conjugated compounds as stealth moieties on the biological behavior of LNP formulations, mRNA expression was examined in vitro using a skeletal muscle cell line (C2C12), a liver cancer cell line (HepG2), and a mouse macrophage cell line (Raw cells). To this end, LNP formulations containing firefly luciferase-encoding mRNA were incubated with the cells at three doses: 12.5 ng/well, 25 ng/well, and 50 ng/well, and luciferase expression was assessed after 24 hours of incubation. Mean fluorescence intensity (MFI) (Figures 27A, C, E) and cell viability (Figures 27B, D, F) were examined by FACS (fluorescence-activated cell sorting) analysis. Figures 27A, C, and E show that luciferase expression was observed in all formulations in the tested cell lines C2C12 (Figures 27A, B), Raw (Figures 27C, D), and HepG2 (Figures 27E, F). Furthermore, the performance and transfection efficiency of amphiphilic OEG conjugate compound-containing LNP formulations depended on the type and concentration of the stealth moiety in the formulation. For VE-AEEA14-AC-containing formulations, reciprocal expression was observed in formulations containing VE-AEEA14-AC, and expression decreased with increasing VE-AEEA14-AC concentration by 2 mol%. Furthermore, for VE-(AEEA)8_AC-containing formulations, expression levels were similar in HY501-VE-(AEEA)8-AC_3 and HY501-VE-(AEEA)8-AC_2. Comparison of VE-(AEEA) ₁₄ -AC and VE-(AEEA)8_AC-containing formulations indicates that formulations with a short stealth moiety exhibit superior performance. The same trend was observed with formulations containing DMA-(AEEA) ₁₄ -AC and DMA-(AEEA)8_AC. Comparison of all tested LNP formulations indicates that the hydrophobic tail type may also play a role in the activity of the formulation, with the following trend observed for expression efficiency: VE≧DMG≧DMA. The same trend was observed with the V09 mRNA-containing formulation (see Example 20).

Example 22 - T Cell Targeting Using LNPs with Various Stealth and Alf Lipids LNPs were formulated with various lipid compositions (cargo: Thy1.1 RNA/Luc RNA/Np proxy Venus 1:1:2 w/w; N/P ratio: 6; lipid mixture: HY501/cholesterol/DSPC/stealth lipid/Alf lipid. The lipid ratio was selected as [47.5/40.5/10/1.8/0.2] for the following combinations of stealth and Alf lipids: C16 PEG2k ceramide/DSPE PEG2k Alfa, DSPE PEG2k/DSPE PEG2k Alfa, DSPE-AEEA14/DSPE-AEEA14-Alfa, or VE-AEEA8/DSPE-AEEA14-Alfa. The lipid ratios for the following stealth lipid and Alf lipid combinations were selected: VE-PEG1k/DSPE-PEG2k-Alfa or VE-AEEA8/DSPE-AEEA14-Alfa. LNPs were post-functionalized with aCD3 VHH x NbAlf ligand (w/w = ligand-to-cargo ratio 0.48; RNA concentration: 0.1 μg/μl). All LNPs had diameters between 100 and 170 nm, as determined by DLS, with PDIs of less than 0.4.

For transfection studies, 10 μl (1000 ng dose) of each formulation was prediluted in 50 μl X-Vivo 15 medium in an ultra-low attachment 96-well plate. ¹⁰ thawed human PBMCs were diluted in 50 μl of 100% coagulation PHS and added to the nanoparticle dilutions. After 30 minutes of incubation (37°C, 5% _CO ), 30 μl of each transfection reaction was transferred to a second ultra-low attachment 96-well plate, and 170 μl of X-Vivo 15 medium + 100 U/ml IL2 was added per well. Cell dilutions were cultured for an additional 18 hours (37°C, 5% _CO ). Specific transfection (Thy1.1) was analyzed by flow cytometry in the following cell types: Per formulation condition tested, the percentage of transfected cells (CD2 negative cells, CD19+ B cells, CD4+ T cells, and CD8+ T cells) within total transfected PBMCs (transfection, y-axis) is given.

As shown in Figure 28, this experiment demonstrates the modular nature of the conjugation approach. As shown for LNPs, ligands can be attached to nanoparticles using Alf lipids with various lipid backbone and spacer types and lengths. Good nanoparticle physicochemical properties (size, PDI, and RNA encapsulation) can be achieved with all stealth/Alf lipid combinations tested. The choice of stealth lipid anchor has an impact on T cell transfection efficiency; DSPE tends to reduce transfection efficiency.

Example 23 - Variation of DMG-AEEA14-AC and C14-AEEA14-AC fractions for the fabrication of nanoparticles using HY501 as an ionizable lipid. The purpose of this example was to test DMG-AEEA14-AC and C14-AEEA14-AC lipids (compound of formula (V-63)) as stealth moieties for the engineering of mRNA nanoparticles using LNP technology with HY501 as the ionizable lipid. LNPs were fabricated by mixing the aqueous RNA phase and the organic lipid phase using a microfluidic device. Various molar fractions of stealth graft lipid (1-3%) were used in the LNP formulation. Molar fractions of the ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of stealth graft lipid) ranging from 47.5:42.5 to X:10 were used, respectively. Luciferase mRNA was used in this example. An N/P ratio of 6 was selected for this experiment. For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these DMG-AEEA14-AC and C14-AEEA14-AC-containing formulations were compared with other PEG-containing formulations, BM_1 and BM_2. Additionally, two LNP formulations containing VE-AEEA14-AC and DMA-AEEA14-AC as stealth moieties were included in this study for comparison purposes. A summary of all formulations used in this study is shown in Table 8.

The size evolution and size distribution of amphiphilic OEG conjugate compound-containing LNPs are shown in Figure 29. As can be seen, the size of amphiphilic OEG conjugate compound-containing LNP formulations decreased with increasing stealth moiety concentration. Furthermore, small particles (d < 100 nm) were obtained using ≥ 2 mol% DMG-AEEA14-AC or C14-AEEA14-AC lipids in the formulation. Meanwhile, all formulations exhibited a PdI of better than 0.3.

Figure 30 shows the zeta potential of all amphiphilic OEG conjugate compound-containing LNPs measured in 0.1x PBS at pH 7. As can be seen, a neutral to slightly negative surface charge was obtained for all formulations.

The RNA accessibility and amount of free RNA in the LNP formulations were measured by the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 31 shows the RNA accessibility of the formulations measured by the RiboGreen assay method (A) or agarose gel electrophoresis method (B). As can be seen, RNA inaccessibility increased with stealth lipid concentration, with high amounts of inaccessible RNA observed in the HY501-DMG-(AEEA) ₁₄ -AC_3 and HY501-C14-(AEEA) ₁₄ -AC_3 formulations. On the other hand, agarose gel electrophoresis images showed that the formulations were nearly free of free RNA. These results from agarose gel measurements indicate that virtually all RNA is bound to the particles of the amphiphilic OEG-conjugated compound-containing LNP formulations.

The formulations were tested for complement activation by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). As can be seen in Figure 32, no complement activation was observed with any of the formulations.

Figure 33 shows the hemolysis analysis after incubation (neutral pH conditions) of the LNP formulations with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, HY501-C14-(AEEA) ₁₄ -AC_1 LNP exhibited a high level of hemolysis, likely due to the large size and low amount of C14-AEEA14-AC in the formulation. Furthermore, negligible hemolysis was observed with the remaining formulations.

To test the effect of various amphiphilic OEG-conjugated compounds as stealth moieties on the biological behavior of LNP formulations, mRNA expression was examined in vitro using a skeletal muscle cell line (C2C12), a liver cancer cell line (HepG2), and a mouse macrophage cell line (Raw cells). To this end, LNP formulations containing firefly luciferase-encoding mRNA were incubated with the cells at three doses: 12.5 ng/well, 25 ng/well, and 50 ng/well, and luciferase expression was assessed after 24 hours of incubation. Mean fluorescence intensity (MFI) (Figure 34A, C, E) and cell viability (Figure 34B, D, F) were examined by FACS (fluorescence-activated cell sorting) analysis. Figures 34A, C, and E show that luciferase expression was observed in all formulations (except for the HY501-C14-(AEEA) ₁₄ -AC_1 LNP in the C2C12 cell line) in the tested cell lines C2C12 (Figures 34A, B), Raw (Figures 34C, D), and HepG2 (Figures 34E, F), respectively. Furthermore, clear OEG mol%-dependent activity was observed upon LNP transfection, while maintaining good viability (>90%). Furthermore, the performance and transfection efficiency of amphiphilic OEG conjugate compound-containing LNP formulations depended on the type of stealth moiety in the formulation. For DMG-AEEA14-AC-containing formulations, the highest expression was observed in formulations containing 2 mol% and 3 mol% DMG-AEEA14-AC. On the other hand, for the C14-AEEA14-AC-containing formulations, the highest expression was observed with the 2 mol% C14-AEEA14-AC-containing formulation. Comparison of the DMG-AEEA14-AC and C14-AEEA14-AC-containing formulations demonstrates the superior performance of the DMG-AEEA14-AC stealth moiety-containing formulation. Furthermore, the transfection efficiency of the DMG-AEEA14-AC-containing formulation was slightly higher than that of the VE-AEEA14-AC and DMA-AEEA14-AC-containing LNPs.

Example 24 - Investigating the Effect of Poly(AEEA) Graft Lipid Length on the Engineering and Biological Behavior of LNP Formulations The purpose of this example was to test poly(AEEA) graft lipids of various lengths as stealth moieties for engineering mRNA nanoparticles using LNP technology containing HY501 as the ionizable lipid. VE-(AEEA)n-AC was used in this example, where n is the number of AEEA repeat units in the hydrophilic block of the stealth graft lipid. VE-(AEEA)n-AC-containing LNPs were fabricated by mixing the RNA aqueous phase and the lipid organic phase using a microfluidic device. Various molar fractions of VE-(AEEA)n-AC graft lipid were used in the LNP formulations. The ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of VE-(AEEA)n-AC graft lipid) were used in molar ratios ranging from 47.5:42.5 to X:10. Luciferase mRNA was used in this study. An N/P ratio of 6 was selected for this experiment. For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these VE-(AEEA)n-AC graft lipid-containing formulations were compared with other PEG-containing formulations, BM_1 and BM_2. A summary of all formulations used in this study is shown in Table 9.

The size evolution and size distribution of amphiphilic VE-(AEEA)n-AC conjugate compound-containing LNPs are shown in Figure 35. The size and size distribution of the BM formulations are also included. As can be seen, the size of amphiphilic VE-(AEEA)n-AC conjugate compound-containing LNP formulations decreased with increasing stealth moiety concentration. Furthermore, small particles (d<100 nm) were obtained in all formulations. On the other hand, all formulations showed a PDI of better than 0.3.

Figure 36 shows the zeta potential of LNPs containing all amphiphilic VE-(AEEA)n-AC conjugate compounds measured in 0.1x PBS at pH 7. Results for the BM formulation are also included. As can be seen, a neutral to slightly negative surface charge was obtained for all formulations. The slightly negative surface charge of the VE-(AEEA)n-AC-containing formulations is likely due to the acetylated functional groups on these molecules.

The RNA accessibility and amount of free RNA in the LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 37 shows the RNA accessibility of the formulations measured using the RiboGreen assay method (A) or agarose gel electrophoresis method (B). As can be seen, high levels of inaccessible RNA were observed in the formulations, regardless of the length and composition of VE-(AEEA)n-AC. On the other hand, agarose gel electrophoresis images showed that free RNA was nearly negligible in the formulations. These results from agarose gel measurements indicate that virtually all RNA is bound to the particles of the LNP formulation containing the amphipathic VE-(AEEA)n-AC conjugate compound.

The complement activation of the formulations was tested by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Figure 38 shows terminal complement complex (SC5b-9) formation after incubation of LNP formulations with human serum. The concentration of SC5b-9 complex formed relative to the control is also included. As can be seen in Figure 38, no or negligible complement activation was observed with all formulations.

Figure 39 shows the hemolysis analysis after incubation (neutral pH conditions) of LNP formulations with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, negligible hemolysis was observed with all other formulations.

To examine the effect of the length of the VE-(AEEA)n-AC as a stealth moiety on the biological behavior of LNP formulations, mRNA expression was examined in vitro using a skeletal muscle cell line (C2C12), a liver cancer cell line (HepG2), and a mouse macrophage cell line (Raw cells). To this end, LNP formulations containing firefly luciferase-encoding mRNA were incubated with cells at three doses: 12.5 ng/well, 25 ng/well, and 50 ng/well, and luciferase expression was assessed after 24 hours of incubation. Mean fluorescence intensity (MFI) (Figures 40A, C, E) and cell viability (Figures 40B, D, F) were examined by FACS (fluorescence-activated cell sorting) analysis. Figure 40 shows that luciferase expression was observed in all formulations in the tested cell lines, with good viability maintained (>80%; with the exception of some formulations at a dose of 50 ng/well in the C2C12 cell line). Furthermore, a comparison of the performance and transfection efficiency of LNP formulations containing VE-(AEEA)n-AC conjugate compounds with various (AEEA)n lengths showed similar levels, with HY501-VE-(AEEA)12-AC_3 and HY501-VE-(AEEA)12-AC_4 LNPs demonstrating the best performance in HepG2 and Raw cell lines.

Example 25 - Investigating the Effect of an OEG Conjugated Compound with Ceramide (C16-Cr-AEEA14-AC) as a Lipid Component on the Engineering and Biological Behavior of LNP Formulations. The purpose of this example was to test an OEG conjugated compound with ceramide as a lipid component (C16-Cr-AEEA14-AC) as a stealth moiety for the engineering of mRNA nanoparticles using LNP technology with HY501 as the ionizable lipid. LNPs were fabricated by mixing an RNA aqueous phase and a lipid organic phase using a microfluidic device. Various molar fractions of the OEG conjugated compound were used in the LNP formulation. The ionizable lipid HY501, helper lipid DSPC, and cholesterol (where X is the fraction of the OEG conjugated compound) were used in respective molar fractions ranging from 47.5:42.5 to X:10. Luciferase mRNA was used in this study. An N/P ratio of 6 was selected for this experiment. For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these LNP formulations containing OEG-conjugated compounds were compared with other PEG-containing BM_1 and BM_2 formulations. VE-(AEEA) ₁₄ -AC-containing LNPs were also used for comparison purposes. A summary of all formulations used in this study is shown in Table 10.

The size evolution and size distribution of amphiphilic OEG conjugate compound-containing LNPs are shown in Figure 41. The size and size distribution of the BM formulations are also included. As can be seen, the size of amphiphilic OEG conjugate compound-containing LNP formulations decreased with increasing stealth moiety concentration. Furthermore, small particles (d<100 nm) were obtained in some formulations. On the other hand, all formulations showed a PDI better than 0.3.

The RNA accessibility and amount of free RNA in the LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis, respectively. Figure 42 shows the RNA accessibility of the formulations measured using the RiboGreen assay (A) or agarose gel electrophoresis (B). As can be seen, a high amount of inaccessible RNA was observed in the C16-Cr-AEEA14-AC-containing LNPs, and the amount of inaccessible RNA increased with increasing stealth lipid concentration. On the other hand, some free RNA was also observed in the HY501-C16-Cr-(AEEA) ₁₄ -AC_1 and HY501-C16-Cr-(AEEA) ₁₄ -AC_1.5 LNPs, with negligible free RNA observed in formulations containing ≥2 mol% C16-Cr-(AEEA) ₁₄ -AC.

The complement activation of the formulations was tested by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Figure 43 shows terminal complement complex (SC5b-9) formation after incubation of LNP formulations with human serum. The concentration of SC5b-9 complex formed relative to the control is also included. As can be seen in Figure 43, no or negligible complement activation was observed with all formulations.

Figure 44 shows the hemolysis analysis after incubation (neutral pH conditions) of the LNP formulation with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, the hemolytic properties of the LNP formulation decrease as the stealth lipid concentration of the formulation increases, likely due to reduced interaction of red blood cells with particles containing high amounts of stealth lipid. Furthermore, small particles interact less with red blood cells than larger particles.

To examine the effect of C16-Cr-AEEA14-AC as a stealth moiety on the biological behavior of the LNP formulation, mRNA expression was examined in vitro using a skeletal muscle cell line (C2C12), a liver cancer cell line (HepG2), and a mouse macrophage cell line (Raw cells). To this end, an LNP formulation containing firefly luciferase-encoding mRNA was incubated with the cells at three doses: 12.5 ng/well, 25 ng/well, and 50 ng/well, and luciferase expression was assessed after 24 hours of incubation. Mean fluorescence intensity (MFI) (Figure 45A, C, E) and cell viability (Figure 45B, D, F) were examined by FACS (fluorescence-activated cell sorting) analysis. Figure 45 shows that low luciferase expression was observed in LNP formulations containing 1 mol% and 1.5 mol% C16-Cr-AEEA14-AC, likely due to the large size of these formulations. Furthermore, luciferase expression in LNP formulations containing 3 mol% C16-Cr-AEEA14-AC was in the range of BM and control LNPs. On the other hand, all formulations maintained good viability (>80%) in the cell lines tested.

Example 26 - Engineering and Biological Feasibility Investigation of Cationic LNPs Containing VE-AEEA14-AC Conjugate Compounds. The purpose of this example was to test VE-AEEA14-AC conjugate compounds for engineering mRNA nanoparticles using LNP technology containing DOTMA and DOTAP as cationic lipids. LNPs were fabricated by mixing the RNA aqueous phase and lipid organic phase using a microfluidic device. Cationic lipids (DOTMA and DOTAP), cholesterol, helper lipids DSPC, and VE-AEEA14-AC were used in molar fractions of 47.5:40.7:10:2, respectively. Luciferase mRNA was used in this study. An N/P ratio of 6 was selected for this experiment. For all LNP formulations, the RNA buffer was 100 mM citrate buffer (CB) pH 4.0, and the dialysis buffer was PBS. The final RNA concentration was 0.05 mg/mL. Furthermore, the physicochemical properties and biological performance of these LNP formulations containing the VE-AEEA14-AC conjugate compound and DOTMA or DOTAP were compared with LNP formulations containing the corresponding ionizable lipids (DODMA and DODAP). Additionally, the BM_1 formulation containing PEG was used as a control in this experiment. A summary of all formulations used in this study is shown in Table 11.

The size evolution and size distribution of LNPs are shown in Figure 46. The size and size distribution of the BM formulations are also included. As can be seen, small particles (d<100 nm) were obtained in all formulations. On the other hand, all formulations showed a PDI greater than 0.25.

Figure 47 shows the zeta potential of all LNPs. Results for the BM formulation are also included. As can be seen, a neutral to slightly negative surface charge was obtained for the BM, DODMA-VE-AEEA14-AC_2, and DODAP-VE-AEEA14-AC_2 formulations. On the other hand, a highly positive surface charge was observed for the DOTMA-VE-AEEA14-AC_2 and DOTAP-VE-AEEA14-AC_2 formulations due to the persistent positive charge of these cationic lipids.

The RNA accessibility and amount of free RNA in the LNP formulations were measured using the RiboGreen assay and agarose gel electrophoresis methods, respectively. Figure 48 shows the RNA accessibility of the formulations measured using the RiboGreen assay method (A) or agarose gel electrophoresis method (B). As can be seen, all LNPs exhibited high amounts of inaccessible RNA. The highest RNA inaccessibility was observed in the DOTMA-VE-AEEA14-AC_2 and DOTAP-VE-AEEA14-AC_2 formulations due to the permanent positive charge of these cationic lipids. On the other hand, no or negligible free RNA was observed in the formulations.

Complement activation of the formulations was tested by in vitro quantification of SC5b-9 levels using the Microvue SC5b-9 Plus ELISA kit (Quidel Co., San Diego, CA, USA). Figure 49 shows terminal complement complex (SC5b-9) formation after incubation of LNP formulations with human serum. The concentration of SC5b-9 complex formed relative to the control is also included. As can be seen in Figure 49, no or negligible complement activation was observed for all formulations. Furthermore, no significant differences in complement activation were observed for LNPs formulated with ionizable and permanent cationic lipids (comparing DODMA and DOTMA or DODAP and DOTAP LNPs).

Figure 50 shows the hemolysis analysis after incubation (neutral pH conditions) of LNP formulations with human whole blood. A hemolysis analysis of the control product is also included. As can be seen, negligible hemolysis was observed with all other formulations.

To examine the effect of persistently charged cationic lipids on the biological behavior of VE-AEEA14-AC-containing LNP formulations, mRNA expression was examined in vitro using a skeletal muscle cell line (C2C12), a liver cancer cell line (HepG2), and a mouse macrophage cell line (Raw cells). To this end, LNP formulations containing firefly luciferase-encoding mRNA were incubated with cells at three doses: 12.5 ng/well, 25 ng/well, and 50 ng/well, and luciferase expression was assessed after 24 hours of incubation. Mean fluorescence intensity (MFI) (Figures 51A, C, E) and cell viability (Figures 51B, D, F) were examined by FACS (fluorescence-activated cell sorting) analysis. Figure 51 shows that expression levels of DOTMA-containing LNPs were significantly lower than those of the corresponding ionizable (DODMA)-based LNPs in all tested cell lines. Furthermore, the expression levels of DODAP and DODAP-containing LNPs were extremely low. Meanwhile, all formulations maintained good viability (>90%) in the tested cell lines.

Example 27 - Preparation, characterization and biological testing of functionalized LNPs containing OEG conjugate compounds
Description of method
Preparation of OEG-conjugated (pAEEA) compound-containing functionalized LNPs. Alfa-tagged lipid particles can be prepared using known aqueous/organic preparation protocols. For the preparation of alfa-tagged lipid nanoparticles, a lipid mixture consisting of HY501, DSPC, cholesterol, DSPE-pAEEA14-alfa peptide, and OEG-conjugated compound was dissolved in organic solvent at a total lipid concentration of 22.8 mM, as shown in Table 12. The lipid mixture was mixed with an aqueous phase containing the relevant cargo (Thy1.1/Luc RNA/DNA 1:1:2 w/w) at an N/P ratio of 12 using a standard syringe pump-based setup and a T-mixing element.

LNPs were post-functionalized with aCD3 VHH x NbAlf ligands (w/w* = ligand-to-cargo ratio 0.48) and diluted with each selected buffer supplemented with sucrose to achieve a final lipid nanoparticle total cargo concentration of 0.1 mg/ml and a final matrix sucrose content of 10% w/v. All formulations were sterile filtered through a 0.22 μm PES filter and stored at -80°C until use.

Characterization of RNA formulations . Particle size was determined by dynamic light scattering using a DynaPro Plate Reader II (Wyatt, Dernbach, Germany). From the measurements, size (Z-average) and polydispersity index (PdI) were calculated by cumulant analysis using Dynamics 7.8.1.3 software. For measurements, samples were diluted 1:10 with water and analyses were performed in triplicate. pH was measured on 150 μL samples after calibrating the device with pH 4, 7, and 10 standards.
Successful cargo incorporation was confirmed via agarose gel electrophoresis.

Freeze-thaw studies Freeze-thaw studies were performed by cycling the formulations at least three times from -20°C or -80°C (overnight) to +25°C (2 hours). Particle size and polydispersity index were measured on the freeze-thawed samples.

For in vitro transfection studies, 5 μl (500 ng dose) of each formulation was prediluted in 50 μl X-Vivo 15 in an ultra-low attachment 96-well plate. 3e5 thawed human PBMCs were diluted in 100% PHS and added to the nanoparticle dilutions. The plate was incubated for 30 minutes (37°C, 5% _CO2 ). 100 μl X-Vivo 15 containing 200 IU/ml IL-2 was added to each well. The plate was incubated for 4 days (37°C, 5% _CO2 ). Transfection (surface Thy1.1 expression) was analyzed by flow cytometry after labeling of samples with cell type-specific antibodies and Thy1.1 detection antibodies.

In vivo administration : 1 μg of each formulation was injected into the thigh muscles of both hind legs of B6-hCD3EDG transgenic mice. Thus, a total dose of 2 μg/mouse was used. 18 hours after LNP injection, the animals were intraperitoneally injected with luciferin and whole-body bioluminescence imaging was performed. Subsequently, the mice were sacrificed, and peripheral lymph nodes, spleens, and livers were isolated. The organs were placed in black well plates containing 500 μl PBS, luciferin was added, and bioluminescence imaging was performed. Lymph nodes and spleens were further processed to obtain single-cell suspensions, which were stained with cell type-specific and Thy1.1 antibodies, and Thy1.1 expression was analyzed by flow cytometry.

Experimental Results: Size and PDI were determined by DLS measurements for functionalized, sterile-filtered lipid nanoparticles stored at a final RNA concentration of 0.1 mg/mL. It was determined that aCD3-VHH-functionalized lipid nanoparticles could be successfully synthesized using pAEEAs-based compounds with various anchoring moieties and various pAEEAs lengths, resulting in particles of approximately 90-100 nm size and PDI ≤ 0.3 (Figure 52).

Successful cargo incorporation was confirmed via agarose gel electrophoresis: no RNA or DNA bands were present in the samples, indicating that the formulations did not carry any free cargo. Furthermore, all RNA and DNA bands were clearly visible when the samples were treated with release solution (control), confirming successful encapsulation of all three cargos (Figure 53).

Figures 54A-E show the results of freezing and storing aCD3-LNPs at -20°C and -80°C, respectively, for at least three freeze/thaw cycles. As can be seen, no significant changes in size or PDI were observed at either freezing temperature tested.

All LNPs tested containing OEG-conjugated (pAEEA) compounds demonstrate RNA transfection of 20-30% of total cells. The percentage of nonspecific transfection of B cells or monocytes was less than 1% within this transfected population for all particles (Figure 55A). Total cell counts after 4 days of incubation demonstrate survival and expansion of all cell subsets (Figure 55B). All particles except C provided an expansion or survival benefit to T cells compared to the no-particle condition (Figure 55B).

The highest luciferase bioluminescence in mice was observed in the popliteal lymph nodes, followed by the inguinal, axillary, and brachial nodes, indicating efficient drainage of particles and transfection into the lymph nodes (Figure 56A). Differences between particles were not statistically significant. Liver accumulation was generally higher in Group E. Groups A and C showed the lowest liver signals (Figure 56A). Groups A, C, and E showed lower T cell transfection in the LNs and spleens of mice than Groups B and D. Group A showed significantly lower T cell transfection in the popliteal LNs than all other groups and in the inguinal LNs than Groups B and D (Figure 56B). Groups B and D showed 1.6% and 0.9% B cell transfection in the popliteal LNs, respectively, and Groups D and E showed 1% and 0.8% PMN transfection, respectively (Table 13). Off-target transfection was less than 1% in the inguinal LNs and spleens.

Claims (159)

A composition comprising: (i) a nucleic acid; (ii) a cationic or cationically ionizable lipid; and (iii) (a) a polymer comprising the following general formula (I): and (b) a polymer conjugate compound comprising one or more hydrophobic chains:
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100. (i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
The composition of claim 1. 3. The composition of claim 1 or 2, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl. The composition of any one of claims 1 to 3, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or methyl. The composition of any of claims 1 to 4, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen. The composition of any one of claims 1 to 5, wherein Y is -CH ₂ - or -(CH ₂ ) ₂ -. The composition of any of claims 1 to 6, wherein Y is -CH ₂ -. The polymer has the following general formula (II):
wherein R ¹ is hydrogen or C _1-8 alkyl.
The composition of any of claims 1 to 7, comprising: The composition of any one of claims 1 to 8, wherein z is 2 to 10, for example, 2 to 7. The composition of any one of claims 1 to 9, wherein z is 2 to 5. The composition of any one of claims 1 to 10, wherein z is 2 or 3. The composition of any one of claims 1 to 11, wherein z is 2. The polymer has the following general formula (III):
wherein R ¹ is hydrogen or C _1-8 alkyl.
13. The composition of any of claims 1 to 12, comprising: 14. The composition of any of claims 8 to 13, wherein R ¹ is hydrogen or methyl. 15. The composition of any of claims 8 to 14, wherein R ¹ is hydrogen. The polymer has the following general formula (IV):
16. The composition of any of claims 1 to 15, comprising: The composition of any one of claims 1 to 16, wherein n is 5 to 50. The composition of any one of claims 1 to 17, wherein n is 5 to 25. Any composition of claims 1 to 18, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. 20. The composition of any of claims 1 to 19, wherein one or more hydrophobic chains are located at the ^X1 or ^X2 end of the polymer. The composition of any one of claims 1 to 20, wherein one or more hydrophobic chains are acyclic, preferably linear, hydrocarbyl groups, more preferably having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The polymer conjugate compound has the following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
22. The composition of any of claims 1 to 21, comprising: (i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
23. The composition of claim 22. The composition of claim 22 or 23, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl. The composition of any of claims 22 to 24, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or methyl. The composition of any of claims 22 to 25, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen. The composition of any of claims 22 to 26, wherein Y is -CH ₂ - or -(CH ₂ ) ₂ -. The composition of any of claims 22 to 27, wherein Y is -CH ₂ -. The polymer conjugate compound has the following general formula (VI) or (VI'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
The composition of any of claims 22 to 28, comprising: The composition of any one of claims 22 to 29, wherein z is 2 to 10, for example, 2 to 7. The composition of any one of claims 22 to 30, wherein z is 2 to 5. The composition of any one of claims 22 to 31, wherein z is 2 or 3. The composition of any one of claims 22 to 32, wherein z is 2. The following general formula (VII) or (VII'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
34. The composition of any of claims 22 to 33, comprising: 35. The composition of any of claims 29 to 34, wherein R ¹ is hydrogen or methyl. 36. The composition of any of claims 29 to 35, wherein R ¹ is hydrogen. The polymer conjugate compound has the following general formula (VIII) or (VIII'):
37. The composition of any of claims 22 to 36, comprising: The composition of any one of claims 22 to 37, wherein n is 5 to 50. The composition of any one of claims 22 to 38, wherein n is 5 to 25. The composition of any one of claims 22 to 39, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. The composition of any of claims 22-40, wherein ^R2 is ^R4 or ^-L1 ( ^R4 ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group; ^L1 is a linker; and p is 1 or 2. ^L1 comprises at least one functionalized moiety such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or the alkylene group is attached to a divalent functionalized moiety at the terminal attached to ^R4 , wherein preferably each monovalent functionalized moiety is hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, ester, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, and/or each divalent functionalized moiety is independently selected from ketal, hemiketal, imide, and amide moieties; and/or each divalent functionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide , carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ](C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(=N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N=)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p comprises a functionalized moiety selected from the group consisting of N(R ²⁶ ) _2-p and [*—C(O)NH](C _1-6 -alkyltriyl)-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; C _1-6 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁵ is selected from the group consisting of C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _1-6 43. The composition of claim 41 or 42, wherein the -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain ^R4 . 44. The composition of any of claims 41-43, wherein L ¹ further comprises at least one additional difunctionalized moiety through which R ² is attached to X ¹ of formula (V) or X ² of formula (V'). At least one further difunctionalized moiety is ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide). , carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide and amide moieties, preferably the group consisting of phosphate, ether, amino, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl and thiocarbonyl, wherein when ^L1 further comprises at least two further difunctionalized moieties, these at least two further difunctionalized moieties are optionally separated from each other by a _C1-6 -alkylene group. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C The composition of any one of claims ₄₁ to 45, wherein the 3,4 _{-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-3 alkyl} , -OH, -CN and -OC _1-3 alkyl; and the C _1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly bonded to another hydrophobic chain R ⁴ . L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)—, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)—, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) ₂ N- and [*-C(O)NH](C _1-6 or L ¹ is (*-)(R ²⁶ )N-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene of [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally halogen, C The composition of any of claims ₄₁ to 46, wherein the C _1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly bonded to another hydrophobic chain R ₄ ^. R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O—, (R ⁴ ) ₂ N—, and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O—, or R ² is (R ⁴ )(R ²⁶ )N—, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl optionally substituted with one or more —OH substituents, and other hydrophobic chains R The composition of any one of claims 22 to 47, wherein the composition is directly bound to ⁴ . 49. The composition of any of claims 22-48, wherein ^R2 is selected from the group consisting of a phosphatidylethanolamine moiety, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or ^R2 is a monoalkylamine moiety. 50. The composition of any of claims 41-49, wherein each ^R4 is independently an acyclic, preferably straight-chain, hydrocarbyl group. 51. The composition of any of claims 41 to 50, wherein each ^R4 is independently a hydrocarbyl group having at least 8 carbon atoms, such as at least 10 carbon atoms or at least 12 carbon atoms. 52. The composition of any of claims 22-51, wherein ^R2 is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and a palmitoylceramide moiety, or ^R2 is a monomyristylamine moiety. R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ^{21 ,} a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ₂₂ and R 23 are independently selected from the group consisting of H, alkyl, _alkenyl , alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , sugars, amino acids, peptides, and members of a targeting pair; and R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C The composition of any of claims 22 to 52, which is substituted with one or more substituents independently selected from the group consisting of _2-6 alkynyl, -COOH, _-NH2 , -NH( _C1-3 alkyl), -N( _C1-3 alkyl) ₂ , a sugar, an amino acid, a peptide and a member of a targeting pair. R ³ is selected from the group consisting of H, C _1-3 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein C each of _{the 1-6} alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ and members of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C 54. The composition of any of claims 22-53, substituted with one or more substituents independently selected from the group consisting of _(1-3 alkyl) ₂ and members of a targeting pair. 55. The composition of any of claims 22-54, wherein R ³ is selected from the group consisting of H, -C(O)(C _1-3 alkyl), -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of _-OH , -SH, halogen, _-CN , -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NH ₂ , -NHCH ₃ , -N(CH ₃ ) ₂ , -C(O)NH 2 , -C(O)NHCH 3 , -C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair. 56. The composition of any of claims 22 to 55, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for said antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor. The polymer conjugate compound has the formula:
[During the ceremony,
n is 5 to 25;
R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl) and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one _or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair;
R ²⁷ is H or a counter cation; and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
57. The composition of any of claims 1 to 56, comprising one of: The composition of claim 57, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. The composition of claim 57 or 58, wherein R ³ is H or —C(O)(C _1-3 alkyl), wherein the C _1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), —SH, —Br, —N ₃ , C _2-6 alkynyl, an antigen, or an antibody. The polymer conjugate compound has the formula:
(Wherein, in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case, —C(O)C ₁₅ H ₃₁ refers to the moiety —C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case, —C(O)C ₁₃ H ₂₇ refers to the moiety —C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case, —C ₁₄ H ₂₉ refers to the moiety —(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case, —C ₁₃ H ₂₇ refers to the moiety —(CH ₂ ) ₁₂ CH ₃ , and in each case, n is 8, 10, or 14.)
60. The composition of any of claims 1 to 59, comprising one of: The polymer conjugate compound has the formula:
(i)
wherein n is 14 and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl);
(ii)
wherein n is 14 and in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl);
(iii)
wherein n is 8, 12, 14, or 16.
61. The composition of any of claims 1 to 60, comprising one of: Any of claims 1-61, wherein the composition is substantially free of lipids or lipid-like substances containing polyethylene glycol (PEG), wherein the PEG has one of at least 30 consecutive ethylene glycol repeating units. Any of the compositions of claims 1 to 62, wherein water is the primary component of the composition and/or the total amount of solvents other than water contained in the composition is less than about 0.5% (v/v). Any of the compositions of claims 1 to 63, wherein the concentration of the nucleic acid in the composition is from about 1 mg/L to about 500 mg/L, for example, from about 1 mg/L to about 100 mg/L, from about 5 mg/L to about 100 mg/L, or from about 10 mg/L to about 100 mg/L. The composition of any one of claims 1 to 64, wherein the cationically ionizable lipid comprises a head group containing at least one tertiary amine moiety. The cationic or cationically ionizable lipid is represented by the formula (X)
[During the ceremony,
one of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)—, —C(═O)NR ^a —, NR ^a C(═O)NR ^a —, —OC(═O)NR ^a — or —NR ^a C(═O)O—; and the other of L ¹⁰ and L ²⁰ is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, SC(═O)—, —NR ^a C(═O)NR ^a -, NR ^a C(═O)NR ^a -, -OC(═O)NR ^a - or -NR ^a C(═O)O- or a direct bond;
G ¹ and G ² are each independently unsubstituted C ₁ -C ₁₂ alkylene or C _2-12 alkenylene;
G ³ is C _1-24 alkylene, C _2-24 alkenylene, C _3-8 cycloalkylene or C _3-8 cycloalkenylene;
R ^a is H or C _1-12 alkyl;
R ³⁵ and R ³⁶ are each independently C _6-24 alkyl or C _6-24 alkenyl;
R ³⁷ is H, OR ⁵⁰ , CN, —C(═O)OR ⁴⁰ , —OC(═O)R ⁴⁰ or —NR ⁵⁰ C(═O)R ⁴⁰ ;
R ⁴⁰ is C _1-12 alkyl;
R ⁵⁰ is H or C _1-6 alkyl; and x is 0, 1, or 2.
66. The composition of any of claims 1 to 65, having the structure of: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof. The cationic or cationically ionizable lipid has the formula (XI):
[During the ceremony,
each of R ₁ and R ₂ is independently R ₅ or -G ₁ -L ₁ -R ₆ , where at least one of R ₁ and R ₂ is -G ₁ -L ₁ -R ₆ ;
each of R ₃ and R ₄ is independently selected from the group consisting of C _1-6 alkyl, C _2-6 alkenyl, aryl, and C _3-10 cycloalkyl;
_R5 and _R6 are each independently an acyclic hydrocarbyl group having at least 10 carbon atoms;
each of G ₁ and G ₂ independently is unsubstituted C _1-12 alkylene or C _2-12 alkenylene;
each of L ₁ and L ₂ is independently selected from the group consisting of —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O) _x —, —S—S—, —C(═O)S—, —SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, —NRaC(═O)NRa—, —OC(═O)NRa— and —NRaC(═O)O—;
Ra is H or C _1-12 alkyl;
m is 0, 1, 2, 3, or 4; and x is 0, 1, or 2.
66. The composition of any of claims 1 to 65, having the structure: Cationic or cationic ionizable lipids include 2,3-dioleyloxy-1-(N,N-dimethylamino)propane (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), and N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP). The composition of any one of claims 1 to 65, comprising dimethylammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DPL14, or a mixture thereof. Any of claims 1 to 68, wherein the cationic or cationically ionizable lipids constitute from about 20 mol% to about 80 mol% of the total lipids present in the composition. The composition of any one of claims 1 to 69 further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably a combination of phospholipids and steroids. The phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine and sphingomyelin, more preferably distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 The composition of claim 70, wherein the phosphatidylethanolamine is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), and diphytanoylphosphatidylethanolamine (DPyPE). The composition of claim 70 or 71, comprising from about 5 mol% to about 30 mol% of the total lipids present in the phospholipid composition. The composition of any one of claims 70 to 72, wherein the steroid comprises a sterol such as cholesterol. Any of claims 70-73, wherein the steroid constitutes about 10 mol% to about 60 mol% of the total lipids present in the composition. Any of the compositions of claims 70-74, wherein the cationic or cationically ionizable lipids constitute about 20 mol% to about 70 mol% of the total lipids present in the composition; the polymer conjugate compounds constitute about 0.5 mol% to about 15 mol% of the total lipids present in the composition; the phospholipids constitute about 5 mol% to about 25 mol% of the total lipids present in the composition; and the steroids constitute about 20 mol% to about 55 mol% of the total lipids present in the composition. The composition of any one of claims 1 to 75, wherein the composition comprises particles dispersed in an aqueous phase, wherein the particles comprise at least a portion of a nucleic acid, at least a portion of a cationic or cationically ionizable lipid, and at least a portion of a polymer-conjugated compound. The composition of claim 76, wherein the particles are selected from lipid nanoparticles (LNPs), liposomes, lipoplexes (LPXs), and mixtures thereof. The composition of claim 76 or 77, wherein the particles constitute at least 50%, preferably at least 75%, and more preferably at least 85% of the nucleic acids present in the composition. The composition of any one of claims 76 to 78, wherein the particles have a size of about 30 nm to about 500 nm. The composition of any one of claims 1 to 79, wherein the nucleic acid is RNA, preferably mRNA. The composition of claim 80, wherein the RNA (1) comprises modified nucleosides in place of uridine, wherein the modified nucleosides are preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (2) has a codon-optimized coding sequence; and/or (3) has a coding sequence with an increased G/C content compared to the wild-type coding sequence. The composition of claim 80 or 81, wherein the RNA comprises at least one, and preferably all, of the following: a 5' cap; a 5' UTR; a 3' UTR; and a polyA sequence. 83. The composition of claim 82, wherein the polyA sequence comprises at least 100 A nucleotides, and wherein the polyA sequence is preferably an interrupted sequence of A nucleotides. The composition of claim 82 or 83, wherein the 5' cap is a cap 1 or cap 2 structure. The composition of any one of claims 80 to 84, wherein the RNA encodes one or more polypeptides, wherein preferably the one or more polypeptides are pharmaceutically active polypeptides and/or contain an epitope for inducing an immune response against an antigen in a subject. The composition of claim 85, wherein the pharmaceutically active polypeptide and/or antigen or epitope is derived from or is a pathogen protein, an immunogenic variant of the protein, or an immunogenic fragment of the protein or an immunogenic variant thereof. A method for delivering a nucleic acid to a cell of a subject, comprising administering to the subject a composition of any one of claims 1 to 86. A method for delivering a therapeutic peptide or protein to a subject, comprising administering to the subject a composition of any one of claims 1 to 86, wherein the nucleic acid encodes the therapeutic peptide or protein. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of any one of claims 1 to 86, wherein delivery of the nucleic acid to the subject's cells is beneficial to treating or preventing the disease or disorder. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of any one of claims 1 to 86, wherein the nucleic acid encodes a therapeutic peptide or protein, and delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder. The method of any one of claims 87 to 90, wherein the subject is a mammal. The method of claim 91, wherein the mammal is a human. (a) a compound of the following general formula (I):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
z is 2 to 24; and n is 1 to 100.
and (b) one or more hydrophobic chains. (i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
94. The polymer conjugate compound of claim 93. 95. The polymer conjugate compound of claim 93 or 94, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or C _1-8 alkyl. 96. The polymer conjugate compound of any of claims 93-95, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or methyl. 97. The polymer conjugate compound of any of claims 93-96, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen. 98. The polymer conjugate compound of any of claims 93-97, wherein Y is -CH ₂ - or -(CH ₂ ) ₂ -. 99. The polymer conjugate compound of any of claims 93-98, wherein Y is -CH ₂ -. The polymer has the following general formula (II):
wherein R ¹ is hydrogen or C _1-8 alkyl.
100. The polymer conjugate compound of any of claims 93-99, comprising: The polymer conjugate compound of any one of claims 93 to 100, wherein z is 2 to 10, for example, 2 to 7. The polymer conjugate compound of any one of claims 93 to 101, wherein z is 2 to 5. The polymer conjugate compound of any one of claims 93 to 102, wherein z is 2 or 3. The polymer conjugate compound of any one of claims 93 to 103, wherein z is 2. The polymer has the following general formula (III):
wherein R ¹ is hydrogen or C _1-8 alkyl.
105. The polymer conjugate compound of any of claims 93-104, comprising: 106. The polymer conjugate compound of any of claims 100-105, wherein R ¹ is hydrogen or methyl. 107. The polymer conjugate compound of any of claims 100-106, wherein R ¹ is hydrogen. The polymer has the following general formula (IV):
108. The polymer conjugate compound of any of claims 93 to 107, comprising: The polymer conjugate compound of any one of claims 93 to 108, wherein n is 5 to 50. The polymer conjugate compound of any one of claims 93 to 109, wherein n is 5 to 25. The polymer conjugate compound of any one of claims 93 to 110, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. 112. The polymer conjugate compound of any of claims 93-111, wherein the one or more hydrophobic chains are located at the ^X1 or ^X2 terminus of the polymer. The polymer conjugate compound of any one of claims 93 to 112, wherein one or more hydrophobic chains are acyclic, preferably linear, hydrocarbyl groups, more preferably having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. The following general formula (V) or (V'):
[During the ceremony,
^X2 and ^X1 together are an optionally substituted amide, an optionally substituted thioamide, an ester, or a thioester;
Y is —CH ₂ —, —(CH ₂ ) ₂ —, or —(CH ₂ ) ₃ —;
^R2 is a moiety comprising one or more hydrophobic chains;
R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, -OR ²⁰ , -SR ²⁰ , halogen, -CN, -N ₃ , -OC(O)R ²¹ , -C(O)R ²¹ , -NR ²² R ²³ , -COOH, -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -COOCH ₃ , -NR ²² R ²³ , -C(O)NR ²² R ²³ , -NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair; R ²⁰ is selected from the group consisting of H, C _1-3 alkyl, and 3- to 6-membered heterocyclyl, wherein each of the C _1-3 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein each of the C _1-6 alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , a sugar, an amino acid, a peptide, and a member of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide, and a member of a targeting pair;
z is 2 to 24; and n is 1 to 100.
114. The polymer conjugate compound of any of claims 93 to 113, comprising: (i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—;
(vi) if X ¹ is —O—, then X ² is —C(O)—;
(vii) if X ¹ is —C(S)—, then X ² is —O—;
(viii) if X ¹ is —O—, then X ² is —C(S)—;
(ix) if X ¹ is —C(O)—, then X ² is —S—; or
(x) if X ¹ is —S—, then X ² is —C(O)—;
where R ¹ is hydrogen or C _1-8 alkyl; preferably
(i) if X ¹ is —C(O)—, then X ² is —NR ¹ —;
(ii) if X ¹ is —NR ¹ —, then X ² is —C(O)—;
(iii) if X ¹ is —C(S)—, then X ² is —NR ¹ —;
(iv) if X ¹ is —NR ¹ —, then X ² is —C(S)—;
(v) if X ¹ is —C(O)—, then X ² is —O—; or
(vi) if X ¹ is —O—, then X ² is —C(O)—;
wherein R ¹ is hydrogen or C _1-8 alkyl;
115. The polymer conjugate compound of claim 114. 116. The polymer conjugate compound of claim 114 or 115, wherein X ¹ is —C(O)— and X ² is —NR ¹ —, where R ¹ is hydrogen or C _1-8 alkyl. 117. The polymer conjugate compound of any of claims 114-116, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen or methyl. 118. The polymer conjugate compound of any of claims 114-117, wherein X ¹ is -C(O)- and X ² is -NR ¹ -, where R ¹ is hydrogen. 119. The polymer conjugate compound of any of claims 114-118, wherein Y is -CH ₂ - or -(CH ₂ ) ₂ -. 120. The polymer conjugate compound of any of claims 114-119, wherein Y is -CH ₂ -. The compound represented by the following general formula (VI) or (VI'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
121. The polymer conjugate compound of any of claims 114-120, comprising: The polymer conjugate compound of any one of claims 114 to 121, wherein z is 2 to 10, for example, 2 to 7. The polymer conjugate compound of any one of claims 114 to 122, wherein z is 2 to 5. The polymer conjugate compound of any one of claims 114 to 123, wherein z is 2 or 3. The polymer conjugate compound of any one of claims 114 to 124, wherein z is 2. The following general formula (VII) or (VII'):
wherein R ¹ is hydrogen or C _1-8 alkyl.
126. The polymer conjugate compound of any of claims 114-125, comprising: 127. The polymer conjugate compound of any of claims 121-126, wherein R ¹ is hydrogen or methyl. 128. The polymer conjugate compound of any of claims 121-127, wherein R ¹ is hydrogen. The following general formula (VIII) or (VIII'):
129. The polymer conjugate compound of any of claims 114-128, comprising: The polymer conjugate compound of any one of claims 114 to 129, wherein n is 5 to 50. The polymer conjugate compound of any one of claims 114 to 130, wherein n is 5 to 25. The polymer conjugate compound of any one of claims 114 to 131, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. 133. The polymer conjugate compound of any of claims 114-132, wherein ^R2 is ^R4 or ^-L1 ( ^R4 ) _p , where each ⁴ is independently a hydrophobic chain such as a hydrocarbyl group; ^L1 is a linker; and p is 1 or 2. ^L1 comprises at least one functionalized moiety such as an alkylene moiety substituted with at least one monovalent functionalized moiety and/or the alkylene group is attached at the terminal attached to ^R4 to a divalent functionalized moiety, wherein preferably each monovalent functionalized moiety is selected from the group consisting of hydroxy, ether, halogen, cyano, azido, nitro, amino, ammonium, ester, carboxyl, thiol (sulfanyl), disulfanyl, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous acid diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, thioisopropyl ... ester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imidamide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemithioate and/or each divalent functionalized moiety is independently selected from ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acid diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, 134. The polymer conjugate compound of claim 133, wherein the aryl group is independently selected from a carboxylate, a carboxyl group ... L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-, [*-NHC(O)] _p (C _1-6 -alkylene)-, [*-C(O)NH] _p (C _1-6 -alkylene)-, [*-S] _p (C _1-6 -alkylene)-, [*-SS] _p (C _1-6 -alkylene)-, [*-S(O) ₂ ] _p (C _1-6 -alkylene)-, [(*-O) _r C(OR ²⁵ ) _3-r ](C _1-6 -alkylene)-, [*-C(OR ²⁵ ) ₂ O] _p (C _1-6 -alkylene)-, [*-C(R ²⁵ )(=N-N(R ²⁶ )C(O)-)] _p (C _1-6 -alkylene)-, [*-C(O)(N(R ²⁶ )-N=)C(R ²⁵ )-] _p (C _1-6 -alkylene)-, [*=C(=N-N(R ²⁶ )C(O)(R ²⁵ ))] _p (C _1-6 -alkylene)-, [*-N(R ²⁶ )N(R ²⁶ )] _p (C _1-6 -alkylene)-, [*=C(=N(OH))] _p (C _1-6 -alkylene)-, [*-OC(R ²⁵ )(R ²⁶ )O] _p (C _1-6 -alkylene)-, *-(3,4-dihydro-2H-chromen-6-yl)-, (*-) _p comprises a functionalized moiety selected from the group consisting of N(R ²⁶ ) _2-p and [*—C(O)NH](C _1-6 -alkyltriyl)-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; C _1-6 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁵ is selected from the group consisting of C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl, and aryl(C _1-6 alkyl); r is an integer from 1 to 2; 3,4-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _1-6 135. The polymer conjugate compound of claim 133 or 134, wherein -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain, ^R4 . 136. The polymer conjugate compound of any of claims 133-135, wherein ^L1 further comprises at least one additional bifunctionalized moiety through which ^R2 is attached to ^X1 of formula (V) or ^X2 of formula (V'). At least one further difunctionalized moiety is an ether, amino, ester, sulfide, disulfide, sulfoxide, sulfone, sulfite, sulfate, phosphate, sulfinamide, sulfonamide, sulfamate, diselenide, sulfurous diamide, acidous diamide, urea, thiourea, carbonyl, thiocarbonyl, orthoester, thioate, dithioate, imidate, imino, imidothioate, thionylamide, carbonate, carbonothioate, carbonodithioate, carbonotrithioate, guanidino(imide) amide), carbamimidate, carbonimidate, carbamate, carbamodithioate, carbonodithioimidate, carbamimidothioate, carbamothioate, carbonimidothioate, acylhydrazone, hydrazine, oxime, acetal, hemiacetal, ketal, hemiketal, imine, imide, and amide moieties, preferably the group consisting of phosphate, imino, sulfate, sulfonamide, urea, thiourea, thioate, dithioate, carbonyl, and thiocarbonyl, wherein when ^L1 further comprises at least two additional difunctionalized moieties, these at least two additional difunctionalized moieties are optionally separated from each other by a _C1-6 -alkylene group. L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NR ²⁶ -, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) _p N(R ²⁶ ) _2-p and [*-C(O)NH](C _1-6 -alkyltriyl)O-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene in [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H, C _1-6 alkyl, aryl and aryl(C _1-6 alkyl); R ²⁷ is H, C The polymer conjugate compound of any of claims ₁₃₃ to 137, wherein the 3,4 _{-dihydro-2H-chromen-6-yl is optionally substituted with one or more substituents selected from the group consisting of halogen, C 1-3 alkyl} , —OH, —CN, and —OC _1-3 alkyl; and the C _1-6 -alkyltriyl is optionally substituted with one or more —OH substituents and is directly bonded to another hydrophobic chain R ⁴ . L ¹ is [*-C(O)O] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)O] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)-, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-OC(O)] _p (C _1-6 -alkylene)-OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)—, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH—, [*-NHC(O)] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)C(O)—, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene), [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )O(C _1-6 -alkylene)NH-, [*-C(O)NH] _p (C _1-6 -alkylene)OP(O)(OR ²⁷ )-O(C _1-6 -alkylene)C(O)-, *-(3,4-dihydro-2H-chromen-6-yl)O-, [*-C(O)O] _p (C _1-6 -alkylene)O-, [*-OC(O)] _p (C _1-6 -alkylene)O-, (*-) ₂ N- and [*-C(O)NH](C _1-6 or L ¹ is (*-)(R ²⁶ )N-, where * represents the point of attachment to R ⁴ ; p is 1 or 2; the C 1-6 -alkylene of [*-C(O)O] _p (C _1-6 -alkylene), [*-OC(O)] _p (C _1-6 -alkylene), [*-NHC(O)] _p (C _1-6 -alkylene) and [*-C(O)NH] _p (C _1-6 -alkylene) is divalent (when p is ₁ ) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl is optionally halogen, C _139. The polymer conjugate compound of any of claims 133 to ₁₃₈ , wherein the C _1-6 -alkyltriyl is optionally substituted with one or more -OH substituents and is directly attached to another hydrophobic chain R ⁴ . R ² is [R ⁴ C(O)O] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)-, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ OC(O)] _p (C _2-3 -alkylene)-OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O—(C _1-3 -alkylene), [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH—, [R ⁴ NHC(O)] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)C(O)-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene), [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )O(C _1-3 -alkylene)NH-, [R ⁴ C(O)NH] _p (C _2-3 -alkylene)OP(O)(OR ²⁷ )-O(C _1-3 -alkylene)C(O)-, (2-R ⁴ -3,4-dihydro-2H-chromen-6-yl)O-, [R ⁴ C(O)O] _p (C _2-3 -alkylene)O-, [*-OC(O)] _p (C _2-3 -alkylene)O—, (R ⁴ ) ₂ N—, and [R ⁴ C(O)NH](C _2-3 -alkyltriyl)O—, or R ² is (R ⁴ )(R ²⁶ )N—, where p is 1 or 2; C _2-3 -alkylene is divalent (when p is 1) or trivalent (when p is 2); R ²⁶ is selected from the group consisting of H and C _1-6 alkyl; R ²⁷ is selected from the group consisting of H and a counter cation; 3,4-dihydro-2H-chromen-6-yl optionally substituted with one or more substituents selected from the group consisting of halogen, C _1-3 alkyl, —OH, —CN, and —OC _1-3 alkyl; and C _2-3 -alkyltriyl optionally substituted with one or more —OH substituents, and other hydrophobic chains R 140. The polymer conjugate compound of any of claims 114 to 139, wherein said polymer conjugate compound is directly bonded to ⁴ . 141. The polymer conjugate compound of any of claims 114-140, wherein ^R2 is selected from a phosphatidylethanolamine, a tocopherol moiety, a diacylglyceride moiety, a dialkylamino moiety, and a ceramide moiety, or ^R2 is a monoalkylamine moiety. 142. The polymer conjugate compound of any of claims 133-141, wherein each ^R4 is independently an acyclic, preferably straight-chain, hydrocarbyl group. 143. The polymer conjugate compound of any of claims 133-142, wherein each ^R4 is independently a hydrocarbyl group having at least 8 carbon atoms, e.g., at least 10 carbon atoms or at least 12 carbon atoms. 144. The polymer conjugate compound of any of claims 114-143, wherein ^R2 is selected from DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine) or POPE (palmitoyloleoylphosphatidylethanolamine), tocopheryl, DMG (1,2-dimyristoylglycerol), DMA (dimyristylamine), and palmitoylceramide moieties, or ^R2 is a monomyristylamine moiety. R ³ is selected from the group consisting of H, C _1-6 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ , a sugar, an amino acid, a peptide, and a member of a targeting pair, wherein the C _1-6 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ^{21 ,} a sugar, an amino acid, a peptide, and a member of a targeting pair; and R ₂₂ and R 23 are independently selected from the group consisting of H, alkyl, _alkenyl , alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ , sugars, amino acids, peptides, and members of a targeting pair; and R ²² and R ²³ are independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl, or R 22 and R 23 together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C The polymer conjugate compound of any of claims 114-144, substituted with one or more substituents independently selected from the group consisting of _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C _1-3 alkyl) ₂ , a sugar, an amino acid, a peptide and a member of a targeting pair. R ³ is selected from the group consisting of H, C _1-3 alkyl, C _2-6 alkynyl, —C(O)R ²¹ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NR ²² R ²³ , —C(O)NR ²² R ²³ , —NR ²² C(O)R ²¹ and members of a targeting pair; and R ²¹ is selected from the group consisting of C _1-6 alkyl and 3- to 6-membered heterocyclyl, wherein C each of _{the 1-6} alkyl and 3- to 6-membered heterocyclyl groups is optionally substituted with one or more substituents independently selected from the group consisting of -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NR ²² R ²³ and members of a targeting pair; and each of R ²² and R ²³ is independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl, or R ²² and R ²³ together with the nitrogen atom to which they are attached may form a heterocyclyl group, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups is optionally substituted with -OH, -SH, halogen, -CN, -N ₃ , C _2-6 alkynyl, -COOH, -NH ₂ , -NH(C _1-3 alkyl), -N(C 146. The polymer conjugate compound of any of claims 114-145, substituted with one or more substituents independently selected from the group consisting of _(1-3 alkyl) ₂ and members of a targeting pair. 147. The polymer conjugate compound of any of claims 114-146, wherein R ³ is selected from the group consisting of H _, —C(O)(C _1-3 alkyl), —NH(C _1-3 alkyl) and —N(C _1-3 alkyl) ₂ and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one or _more substituents independently selected from the group consisting of —OH, _—SH , halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH 3 , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH 2 , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair. 148. The polymer conjugate compound of any of claims 114 to 147, wherein the targeting pair is selected from the following pairs: maleimide-thiol; thiol-halogenated (especially brominated) alkyl; azide-alkyne (especially in copper(I) catalyzed reactions); conjugated diene-substituted alkene (dienophile) (especially in Diels-Alder reactions); antigen-antibody specific for the antigen; biotin-streptavidin; biotin-avidin; biotin-neutravidin; folate-folate receptor; transferrin-transferrin receptor; aptamer-molecule for which the aptamer is specific; arginine-glycine-aspartic acid (RGD) peptide-α _v β ₃ integrin; asparagine-glycine-arginine (NGR) peptide-aminopeptidase N; galactose-asialoglycoprotein receptor. The following formula:
[During the ceremony,
n is 5 to 25;
R ³ is selected from the group consisting of H, —C(O)(C _1-3 alkyl) and members of a targeting pair, wherein the C _1-3 alkyl group is optionally substituted with one _or more substituents independently selected from the group consisting of —OH, —SH, halogen, —CN, —N ₃ , C _2-6 alkynyl, —COOH, —COOCH ₃ , —NH ₂ , —NHCH ₃ , —N(CH ₃ ) ₂ , —C(O)NH ₂ , —C(O)NHCH 3 , —C(O)NH(CH ₂ ) ₂ NH ₂ and members of a targeting pair;
R ²⁷ is H or a counter cation; and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case -C(O)C ₁₅ H ₃₁ refers to the moiety -C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case -C(O)C ₁₃ H ₂₇ refers to the moiety -C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case -C ₁₃ H ₂₇ refers to the moiety -(CH ₂ ) ₁₂ CH ₃ , and in each case -C(O)C ₁₇ H ₃₃ refers to the moiety -cis-C(O)(CH ₂ ) ₇ -CH═CH-(CH ₂ ) ₇ CH ₃ (oleoyl).
149. The polymer conjugate compound of any of claims 93-148, having the formula: The polymer conjugate compound of claim 149, wherein n is 7 to 16, for example 7 to 14, preferably 8, 10, 12, 14, or 16. 151. The polymer conjugate compound of claim 149 or ₁₅₀ , wherein ^R3 is H or -C(O)( _C1-3 alkyl), wherein the _C1-3 alkyl group is optionally substituted with one substituent selected from the group consisting of 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl (maleimidyl), -SH, -Br, _-N3 , and C2-6 alkynyl. The following formula:
(Wherein, in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl), in each case, —C(O)C ₁₅ H ₃₁ refers to the moiety —C(O)(CH ₂ ) ₁₄ CH ₃ (palmitoyl), in each case, —C(O)C ₁₃ H ₂₇ refers to the moiety —C(O)(CH ₂ ) ₁₂ CH ₃ (myristoyl), in each case, —C ₁₄ H ₂₉ refers to the moiety —(CH ₂ ) ₁₃ CH ₃ (myristyl), in each case, —C ₁₃ H ₂₇ refers to the moiety —(CH ₂ ) ₁₂ CH ₃ , and in each case, n is 8, 10, 12, 14, or 16.)
152. The polymer conjugate compound of any of claims 93-151, having the formula: The following formula:
(i)
wherein n is 14 and in each case -C(O)C ₁₇ H ₃₅ refers to the moiety -C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl);
(ii)
wherein n is 14 and in each case -C ₁₄ H ₂₉ refers to the moiety -(CH ₂ ) ₁₃ CH ₃ (myristyl);
(iii)
wherein n is 8, 12, 14, or 16.
153. The polymer conjugate compound of any of claims 93-152, having the formula: (a) a polymer conjugate compound of any one of claims 93 to 153 comprising a member of a targeting pair; and (b) a conjugate of a compound comprising another member of the targeting pair. The conjugate of claim 154, wherein the compound comprising the other member of the targeting pair further comprises a sugar, an amino acid, a peptide (e.g., an antigen or epitope), or an antibody. The following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); m1 and m2 are each independently 1, 2, 3, 4, or 5; and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. The following expression
(i)
wherein m1 is 2, 3, or 4, preferably 2; and m2 is 2, 3, or 4, preferably 2;
(ii)
[In the formula, m1 and m2 each independently represent 1, 2, or 3, and preferably m1 is 1 and m2 is 2, or m1 is 2 and m2 is 1.]
157. The conjugate of claim 156, having one of: The following formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
or a salt thereof. The polymer conjugate compound comprising a member of a targeting pair has the formula:
wherein n is 5 to 25, preferably 8, 10, 12, 14, or 16; in each case, —C(O)C ₁₇ H ₃₅ refers to the moiety —C(O)(CH ₂ ) ₁₆ CH ₃ (stearoyl); and Pept is an antigen or an antibody specific for said antigen.
having
The conjugate of claim 154, wherein the compound comprising the other member of the targeting pair is (i) an antibody specific to the antigen if Pept is the antigen; or (ii) a compound comprising the antigen if Pept is an antibody specific to the antigen; and the polymer conjugate compound comprising a member of the targeting pair is conjugated to the compound comprising the other member of the targeting pair via interaction of (1) the antibody specific to the antigen and (2) the antigen.