CN121463947A

CN121463947A - Delivery of polynucleotides from lipid nanoparticles comprising RNA and ionizable lipids

Info

Publication number: CN121463947A
Application number: CN202480041069.0A
Authority: CN
Inventors: M·莫萨赫博; S·帕特尔; E·特洛伊; Y·法拉; M·高夫; A·豪; S·巴尔穆里; S·阿迪卡里
Original assignee: Sail Biopharmaceutical Co ltd
Current assignee: Sail Biopharmaceutical Co ltd
Priority date: 2023-04-19
Filing date: 2024-04-18
Publication date: 2026-02-03
Also published as: WO2024220625A1

Abstract

本文提供了一种用于将RNA组合物递送至受试者以在所述受试者体内产生蛋白质或肽的方法，包括向所述受试者施用包含编码蛋白质或肽的多核苷酸的RNA组合物，所述多核苷酸被配制在以下结构内：（a）多个包含合成结构脂质和可电离脂质的脂质纳米颗粒（LNP）或（b）包含天然脂质和可电离脂质的脂质重构天然信使包（LNMP）。本文还提供了用于治疗与胃肠道、胃、小肠或大肠、肠系膜淋巴结、胰腺、结肠或直肠、盲肠和/或脾相关的疾病或障碍的方法，包括经口或经肠施用本文所述的RNA组合物。This document provides a method for delivering an RNA composition to a subject to generate a protein or peptide in the subject, comprising administering to the subject an RNA composition comprising a polynucleotide encoding a protein or peptide, said polynucleotide being formulated within: (a) a plurality of lipid nanoparticles (LNPs) comprising synthetic structural lipids and ionizable lipids or (b) a lipid reconstructed natural messenger package (LNMP) comprising natural lipids and ionizable lipids. This document also provides a method for treating diseases or disorders associated with the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph nodes, pancreas, colon or rectum, cecum, and/or spleen, comprising oral or enteral administration of the RNA composition described herein.

Description

Delivery of polynucleotides from lipid nanoparticles comprising RNA and ionizable lipids

Cross Reference to Related Applications

The present application claims priority from U.S. provisional application No. 63/460,586 filed on month 4 and 19 of 2023, U.S. provisional application No. 63/502,522 filed on month 5 and 16 of 2023, and U.S. provisional application No. 63/547,630 filed on month 11 and 7 of 2023, all of which are incorporated herein by reference in their entirety.

Background

The use of polynucleotides as therapeutic agents is an emerging field. Thus, there is a need to develop oral and enteral polynucleotide delivery systems for more efficient, easy large-scale and stable delivery of RNA therapeutics that will help patients follow the treatment. There is also a need for delivery methods that target the gastrointestinal tract as well as the surrounding immune system.

Disclosure of Invention

In one aspect, provided herein is a method for delivering an RNA composition to a subject to produce a protein or peptide in the subject. The method comprises administering (e.g., by oral, enteral, or intravenous delivery) to the subject an RNA composition comprising one or more polynucleotides encoding a protein or peptide (e.g., mRNA or circRNA) formulated within (a) a plurality of Lipid Nanoparticles (LNPs) comprising synthetic structural lipids and ionizable lipids, or (b) a Complex Lipid Particle (CLP) comprising a natural lipid and an ionizable lipid, such as a lipid reconstituted natural messenger package (LNMP).

The ionizable lipid has two or more of the following listed characteristics:

(i) At least one ionizable amine;

(ii) At least three lipid tails, wherein each lipid tail is at least six carbon atoms in length;

(iii) pKa from about 4.5 to about 7.5;

(iv) An ionizable amine and a heteroorganic group separated by a chain of at least two atoms, and

(V) The ratio N to P is at least 3.

In some embodiments, the RNA composition is administered orally or enterally. In some embodiments, the RNA composition is administered systemically (e.g., intravenously).

In another aspect, provided herein is a method for delivering an RNA composition to a lymphatic transport system and bypassing the liver. The method comprises administering to a subject an RNA composition comprising one or more polynucleotides (e.g., mRNA or circRNA) encoding one or more polypeptides formulated within a structure comprising an LNP of synthetic structural lipids and ionizable lipids, or a CLP comprising natural lipids and ionizable lipids, e.g., LNMP. The ionizable lipid has two or more of the following listed characteristics:

(i) At least one ionizable amine;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

In another aspect, provided herein are methods of treating diseases or disorders associated with the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph nodes, pancreas, colon or rectum, cecum and/or spleen. The method comprises orally administering an RNA composition having one or more polynucleotides encoding one or more polypeptides. The one or more polynucleotides are formulated within a plurality of Lipid Nanoparticles (LNPs) comprising synthetic structural lipids and ionizable lipids, or CLPs comprising natural lipids and ionizable lipids, such as lipid-reconstituted natural messenger package (LNMP). The ionizable lipid has two or more of the following listed characteristics:

(i) At least one ionizable amine;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

In some embodiments, the disease or disorder is pancreatitis, inflammatory Bowel Disease (IBD), crohn's disease, colorectal cancer, or ulcerative colitis.

In another aspect of the invention, provided herein is a method for delivering an antibody to a subject, comprising:

Systemically administering to a subject an RNA composition comprising one or more polynucleotides encoding antibodies (e.g., mRNA or circRNA) formulated within a structure comprising a Complex Lipid Particle (CLP) comprising a natural lipid and an ionizable lipid, such as a lipid reconstituted natural messenger package (LNMP), wherein the ionizable lipid has two or more of the following listed characteristics:

(i) At least 2 ionizable amines;

(ii) At least 3 lipid tails, wherein each lipid tail is at least six carbon atoms in length;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

Systemically administering to a subject an RNA composition comprising one or more polynucleotides encoding antibodies (e.g., mRNA or circRNA) formulated within a plurality of Lipid Nanoparticles (LNPs) comprising synthetic structural lipids and ionizable lipids, wherein the ionizable lipids have two or more of the following listed characteristics:

(i) At least 2 ionizable amines;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

In another aspect, provided herein is an oral vaccine composition (e.g., for viral or bacterial infection or for anti-tumor) comprising an RNA composition having one or more polynucleotides (e.g., mRNA or circRNA) encoding one or more polypeptides formulated within (a) a plurality of Lipid Nanoparticles (LNPs) comprising synthetic structural lipids and comprising ionizable lipids, or

(B) Lipid reconstitution natural messenger package (LNMP) comprising natural lipids and ionizable lipids. The ionizable lipid has two or more of the following listed characteristics:

(i) At least 1 ionizable functional amine;

(iii) pKa from about 4.5 to about 7.5;

(iv) An ionizable amine functional group and a heteroorganic functional group separated by a chain of at least two atoms, and

(V) The ratio of N to P is at least 3, wherein the mRNA composition is formulated in an oral dosage form. In some embodiments, the polypeptide is an antigenic polypeptide derived from an infectious pathogen that causes a viral or bacterial infection. In some embodiments, the antigenic polypeptide is a coronavirus.

All embodiments discussed below apply to all aspects above.

In some embodiments, the polynucleotide is mRNA or circRNA. In some embodiments, the mRNA or circRNA is derived from (a) a DNA molecule, or (b) an RNA molecule, wherein T is replaced with U.

In some embodiments, the polynucleotide encodes a protein, peptide, or polypeptide comprising an antibody. In some embodiments, the antibody is a therapeutic agent. In one embodiment, the antibody is a TNF inhibitor or a PCSK9 inhibitor. In one embodiment, an RNA composition comprising one or more polynucleotides encoding the antibody (e.g., mRNA or circRNA) is administered at least once, optionally two or more times.

In some embodiments, the in vivo production of the protein or peptide (or polypeptide) occurs in the stomach, small intestine, mesenteric lymph node, pancreas, colon, cecum and/or spleen of the subject. In some embodiments, administration of the RNA composition results in expression of the protein or the peptide (or polypeptide) being detectable in one or more organs of the subject at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In some embodiments, the organ is distributed along a transport path of the alimentary canal, and the RNA composition reaches the organ through a lymphatic transport system.

In some embodiments, the protein or the peptide (or polypeptide) encoded by the polynucleotide (e.g., polyribonucleotide, mRNA, or circRNA) is detectable in the subject's mesenteric lymph node, pancreas, stomach, colon, spleen, and/or small intestine (e.g., villus, peyer's Patch) at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration. In some embodiments, the protein or the peptide (or polypeptide) encoded by the polynucleotide is undetectable in the liver of the subject at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

In some embodiments, the RNA composition is formulated within a plurality of Lipid Nanoparticles (LNPs) comprising a synthetic structural lipid and an ionizable lipid.

In some embodiments, the RNA composition is formulated within an LNMP comprising natural lipids and ionizable lipids.

In some embodiments, the RNA composition is formulated within CLP. In some embodiments, the CLP is an LNMP. Thus, all embodiments described below for the features associated with LNMP and LNMP formulations apply to CLP and CLP formulations.

In some embodiments, the polynucleotide is encapsulated by the LNP or LNMP. In some embodiments, the polynucleotide is embedded on the surface of the LNP or LNMP. In some embodiments, the polynucleotide is conjugated to the surface of the LNP or LNMP.

In some embodiments, the RNA composition is administered to the subject in a delayed release pharmaceutical dosage form comprising (a) a therapeutically effective amount of a polynucleotide (e.g., mRNA or circRNA), (b) a bile salt or cholic acid, and (c) at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.

In some embodiments, the RNA composition is administered in the form of a capsule (e.g., a starch capsule, a cellulose capsule, a hard gelatin capsule, or a soft gelatin capsule). In some embodiments, the RNA composition is administered in the form of a tablet or caplet (caplet). In some embodiments, the capsule, tablet or caplet comprises an enteric coating. In some embodiments, the RNA composition is administered in the form of a plurality of particles, microparticles, beads, pellets, or a mixture thereof.

In some embodiments, the RNA composition comprises:

One or more polynucleotides encoding one or more polypeptides (e.g., mRNA or circRNA) formulated within a CLP (e.g., lipid reconstituted natural messenger package (LNMP)) comprising natural lipids and ionizable lipids,

Wherein the ionizable lipid has two or more of the following listed characteristics:

(i) At least one ionizable amine functional group;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

In some embodiments, the ionizable lipid may be 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol )（1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl) (2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol)）（C12-200）、MD1（cKK-E12）、OF2、EPC、ZA3-Ep10、TT3、LP01、5A2-SC8、, SM-102 (lipid H), or ALC-315. In one embodiment, the ionizable lipid included is C12-200.

In some embodiments, the ionizable lipid involved is

,

Wherein R is a C ₈-C₁₄ alkyl group.

In all these aspects, the ionizable lipid may be selected from one of the following group of compounds:

i) And A compound of (a), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:

Each a is independently C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally substituted with heteroatoms or with OH, SH or halogen;

Each B is independently C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally substituted with heteroatoms or with OH, SH or halogen;

Each X is independently a biodegradable moiety, and

W is

;

Or (b)Wherein:

R ₅ is OH, SH or NR ₁₀R₁₁;

Each R ₆ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, or cycloalkyl;

Each R ₇ and each R ₈ are independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH, SH, or NR ₁₀R₁₁, wherein each R ₁₀ and R ₁₁ are independently H, C ₁-C₃ alkyl, or R ₁₀ and R ₁₁ together form a heterocycle;

Each s is independently 1, 2, 3, 4, or 5;

Each u is independently 1, 2, 3, 4, or 5;

t is 1, 2, 3, 4 or 5;

Each Z is independently absent, O, S or NR ₁₂, wherein R ₁₂ is H, C ₁-C₇ branched or unbranched alkyl, or C ₂-C₇ branched or unbranched alkenyl, and

Q is O, S or NR ₁₃, wherein each R ₁₃ is H or C ₁-C₅ alkyl;

ii) the method comprises the steps of A compound of (a), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:

is a cyclic or heterocyclic moiety;

Y is alkyl, hydroxy, hydroxyalkyl or ;

A is absent, -O-, -N (R ⁷) -, -O-alkylene-, -alkylene -O-、-OC(O)-、-C(O)O-、-N(R⁷)C(O)-、-C(O)N(R⁷)-、-N(R⁷)C(O)N(R⁷)-、-S-、-S-S-, or a divalent heterocyclic ring;

Each of X and Z is independently absent, -O-, -CO-, -N (R ⁷) -, -O-alkylene-, -alkylene-O-, -OC (O) -, -C (O) O-, -N (R ⁷)C(O)-、-C(O)N(R⁷) -, or-S-;

Each R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, hydroxyalkyl, or aminoalkyl;

each M is independently a biodegradable moiety;

Each of R ₃₀、R₄₀、R₅₀、R₆₀、R₇₀、R₈₀、R₉₀、R₁₀₀、R₁₁₀ and R ₁₂₀ is independently H, C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

each of l and m is an integer from 1 to 10;

t1 is an integer from 0 to 10, and

W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl, and

Iii) AndOr (b)A compound of (a), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing, wherein:

R ₂₀ and R ₃₀ are each independently H, C ₁-C₅ branched or unbranched alkyl, or C ₂-C₅ branched or unbranched alkenyl, or R ₂₀ and R ₃₀ together with the adjacent N atom form a3 to 7 membered ring, optionally substituted with R ^a;

R ^a is H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH or SH;

Each R ₁ and each R ₂ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, OH, halogen, SH or NR ₁₀R₁₁, or

R ₁ and R ₂ together form a ring;

Each R ₁₀ and R ₁₁ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, or R ₁₀ and R ₁₁ together form a heterocycle;

n is 0, 1, 2, 3 or 4;

Y is O or S;

Z is absent, O, S, or N (R ₁₂), wherein each R ₁₂ is independently H, C ₁-C₇ branched or unbranched alkyl, or C ₂-C₇ branched or unbranched alkenyl, provided that when Z is present, adjacent R ₁ and R ₂ cannot be OH, NR ₁₀R₁₁, or SH;

v is 0, 1, 2, 3 or 4;

y is 0, 1, 2, 3 or 4;

Each a is independently a C ₁-C₁₆ branched or unbranched alkyl group, or a C ₂-C₁₆ branched or unbranched alkenyl group, optionally interrupted by one or more heteroatoms or optionally substituted with OH, SH or halogen;

Each B is independently a C ₁-C₁₆ branched or unbranched alkyl group, or a C ₂-C₁₆ branched or unbranched alkenyl group, optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen, and

Each X is independently a biodegradable moiety, and

Iv) a lipid comprising at least one head group and at least one tail group of formula (TI) or (TI'), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

Or (b)

,

Wherein:

e are each independently -OC(O)-、-C(O)O-、-N(R⁷)C(O)-、-C(O)N(R⁷)-、-C(O-R₁₃)-O-、-C(O)O(CH₂)_r-、-C(O)N(R⁷)(CH₂)_r-、-S-S- or-C (O-R ₁₃)-O-(CH₂)_r -, wherein each R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl;

R ₁₃ is branched or unbranched C ₃-C₁₀ alkyl;

r is 1, 2, 3, 4 or 5;

Each R ^a is independently C ₁-C₅ alkyl, C ₂-C₅ alkenyl or C ₂-C₅ alkynyl;

u1 and u2 are each independently 0,1, 2,3, 4, 5, 6 or 7;

each R ^t is independently H, C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

Represents a bond linking the tail group and the head group, and

Wherein the lipid has a pKa of about 4 to about 8.

In some embodiments, the ionizable lipid is of the formula

A compound of group i) represented, pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein:

Each R ₁ and each R ₂ is independently H, C ₁-C₃ branched or unbranched alkyl, OH, halogen, SH or NR ₁₀R₁₁, or

Each R ₁ and each R ₂ independently form a ring together with the carbon atoms to which they are attached;

Each R ₁₀ and R ₁₁ is independently H, C ₁-C₃ branched or unbranched alkyl, or R ₁₀ and R ₁₁ together form a heterocycle;

Each R ₃ and each R ₄ are independently H, C ₂-C₁₄ branched or unbranched alkyl (e.g., C ₃-C₁₀ branched or unbranched alkyl), or C ₃-C₁₀ branched or unbranched alkenyl, provided that at least one of R ₃ and R ₄ is not H;

Each X is independently a biodegradable moiety;

Each q is independently 2,3, 4 or 5;

V is a branched or unbranched C ₂-C₁₀ alkylene, C ₂-C₁₀ alkenylene, C ₂-C₁₀ alkynylene, or C ₂-C₁₀ heteroalkylene, optionally substituted with one or more OH, SH, and/or halogen groups;

Each R ₇ and each R ₈ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH, SH, (CH ₂)_vR₁₇ or NR ₁₀R₁₁, wherein each v is independently 0, 1,2,3,4 or 5, and R ₁₇ is OH, SH or N (CH ₃)₂; and

Each m is independently 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, V is a branched or unbranched C ₂-C₃ alkylene group, and each R ₆ is independently H or methyl.

In some embodiments, the ionizable lipid is of the formulaA compound of group i) represented, pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, wherein:

Each X is independently a biodegradable moiety;

Each s is independently 1, 2, 3, 4, or 5;

T is-NHC (O) O-, -OC (O) NH-; or a divalent heterocyclic ring optionally substituted with one or more- (CH ₂)_vOH、-(CH₂)_vSH、-(CH₂)_v -halogen groups),

Each R ₇ and each R ₈ are independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH, SH, (CH ₂)_vR₁₇ or NR ₁₀R₁₁, wherein R ₁₇ is OH, SH or N (CH ₃)₂;

each v is independently 0,1, 2, 3, 4 or 5, and

Each m is independently 1,2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, T is a divalent piperazine or a divalent dioxopiperazine.

In some embodiments, in the formulas of group i) above, X is-OCO-, -COO-, -NHCO-or-CONH-.

In some embodiments, the ionizable lipid is a compound of group ii) represented by the formula:

、 Or (b) Wherein:

t1 is an integer from 0 to 10;

W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl;

each M is independently a biodegradable moiety;

Each m1 is independently an integer from 3 to 6,

Each l1 is independently an integer from 4 to 8,

M2 and l2 are each independently an integer from 0 to 3,

R ₈₀ and R ₉₀ are each independently unsubstituted C ₅-C₈ alkyl or alkenyl, or R ₈₀ is H or unsubstituted C ₁-C₄ alkyl or alkenyl, and R ₉₀ is unsubstituted C ₅-C₁₁ alkyl or alkenyl, and

R ₁₁₀ and R ₁₂₀ are each independently unsubstituted C ₅-C₈ alkyl or alkenyl, or R ₁₁₀ is H or unsubstituted C ₁-C₄ alkyl or alkenyl, and R ₁₂₀ is unsubstituted C ₅-C₁₁ alkyl or alkenyl. In some embodiments, M is-OC (O) -or-C (O) O-;

Is that

Or (b),

Each R ^c is independently H or C ₁-C₃ alkyl, and

Each t1 is independently 1, 2, 3 or 4.

In some embodiments, the ionizable lipid is a compound of group iii), wherein R ₁ and R ₂ are each H, or each R ₁ is H and one of the R ₂ variables is OH, and X is-OC (O) -or-C (O) O-. In some embodiments, the ionizable lipid is a compound of group III) represented by formula III, wherein R ₂₀ and R ₃₀ are each independently H or C ₁-C₃ branched or unbranched alkyl, or R ₂₀ and R ₃₀ together with the adjacent N atom form a3 to 7 membered ring, optionally substituted with R ^a, R ^a is H or OH, Z is absent, S, O or NH, and N is 0,1 or 2. In some embodiments, the ionizable lipid is a compound of group iii) represented by formula V.

In some embodiments, the ionizable lipid is a compound of group iv), wherein the lipid comprises at least a head group and at least a tail group, wherein:

The tail group has the structure of formula (TI) or formula TI':

Or (b) And (C) sum

The head group has the structure of one of the following formulas:

i),

Wherein:

R ₂₀ and R ₃₀ are each independently H, C ₁-C₅ branched or unbranched alkyl or C ₂-C₅ branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl, or

R ₂₀ and R ₃₀ together with the adjacent N atoms form a3 to 7 membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl or cycloalkyl groups;

Each of R ₁ and R ₂ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, OH, halogen, SH or NR ₁₀R₁₁, or R ₁ and R ₂ together form a ring;

Each of R ₁₀ and R ₁₁ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, or R ₁₀ and R ₁₁ together form a heterocycle;

n is 0, 1,2,3 or 4, and

Z is absent, O, S or NR ₁₂ wherein R ₁₂ is H or C ₁-C₇ branched or unbranched alkyl, provided that when Z is present, adjacent R ₁ and R ₂ cannot be OH, NR ₁₀R₁₁ or SH;

ii)

Wherein:

r ₁ is H, C ₁-C₃ alkyl, OH, halogen, SH, or NR ₁₀R₁₁;

R ₂ is OH, halogen, SH or NR ₁₀R₁₁, or R ₁ and R ₂ may together form a ring;

R ₁₀ and R ₁₁ are each independently H or C ₁-C₃ alkyl, or R ₁₀ and R ₁₁ may together form a heterocycle;

R ₂₀ and R ₃₀ are each independently H, C ₁-C₅ branched or unbranched alkyl, C ₂-C₅ branched or unbranched alkenyl, or R ₂₀ and R ₃₀ can together form a ring, and

V and y are each independently 1,2, 3 or 4;

iii)

Wherein W is

Or (b),

Wherein the method comprises the steps of

R ₅ is OH, SH, (CH ₂)_s OH or NR ₁₀R₁₁;

Each R ₇ and R ₈ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, (CH ₂)_vOH、(CH₂)_vSH、(CH₂)_sN(CH₃)₂ or NR ₁₀R₁₁, wherein each R ₁₀ and R ₁₁ is independently H or C ₁-C₃ alkyl, or R ₁₀ and R ₁₁ together form a heterocycle, or R ₇ and R ₈ together form a ring;

Each R ₂₀ is independently H or C ₁-C₃ branched or unbranched alkyl;

R ₁₄ is a heterocycle, NR ₁₀R₁₁、C(O)NR₁₀R₁₁、NR₁₀C(O)NR₁₀R₁₁ or NR ₁₀C(S)NR₁₀R₁₁ wherein each R ₁₀ and R ₁₁ is independently H, C ₁-C₃ alkyl, C ₃-C₇ cycloalkyl, C ₃-C₇ cycloalkenyl, optionally substituted with one or more NH and/or oxo groups, or R ₁₀ and R ₁₁ together form a heterocycle;

R ₁₆ is H, =o, =s or CN;

Each of s, u and t is independently 1, 2, 3, 4 or 5;

each v is independently 0,1, 2, 3, 4, or 5;

Each Y is a divalent heterocyclic ring;

Each Z is independently absent, O, S, or NR ₁₂, wherein R ₁₂ is H, C ₁-C₇ branched or unbranched alkyl, or C ₂-C₇ branched or unbranched alkenyl;

Q is O, S, CH ₂ or NR ₁₃, wherein each R ₁₃ is H or C ₁-C₅ alkyl;

V is a branched or unbranched C ₂-C₁₀ alkylene, C ₂-C₁₀ alkenylene, C ₂-C₁₀ alkynylene or C ₂-C₁₀ heteroalkylene, optionally substituted with one or more OH, SH and/or halogen groups, and

T is-NHC (O) O-, -OC (O) NH-or a divalent heterocycle, and

iv)

Wherein:

is a cyclic or heterocyclic moiety;

y is alkyl, hydroxy, hydroxyalkyl, Or (b);

A is absent, -O-, -N (R ⁷) -, -O-alkylene-, -alkylene-O-, -OC (O) -, -C (O) O-, -N (R ⁷)C(O)-、-C(O)N(R⁷)-、-N(R⁷)C(O)N(R⁷) -, -S-or-S-S-;

Each of X and Z is independently absent, -O-, -C (O) -, -N (R ⁷) -, -O-alkylene-, -alkylene-O-, -OC (O) -, -C (O) O-, -N (R ⁷)C(O)-、-C(O)N(R⁷) -or-S-;

Each R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxy, alkoxy, hydroxyalkyl, alkylamino, alkylaminoalkyl, or aminoalkyl;

t is 0,1, 2 or 3;

t1 is an integer from 0 to 10, and

Wherein the lipid has a pKa of about 4 to about 8.

In some embodiments, the ionizable lipid is a compound of group iv), and wherein at least one tail group of the lipid has one of the following formulas:

And Wherein:

Each R ₇ is independently H or methyl;

R ^b is independently in each occurrence H or C ₁-C₄ alkyl;

u1 and u2 are each independently 0,1, 2,3, 4, 5, 6 or 7, and

U3 and u4 are each independently 0,1, 2,3, 4, 5, 6 or 7, and

The head group has the structure of one of the following formulas:

i) Wherein m is 1, 2, 3,4, 5, 6, 7 or 8;

ii)

iii) A kind of electronic device

iv)。

In some embodiments, at least one tail group has the structure of formula (TII), (TIV), (TV), (TII ') and/or (TIII'), wherein u1 is 3-5, u2 is 0-3, u3 and u4 are each independently 1-7, and R ^a is each independently methyl.

In some embodiments, the tail group has the structure of formula (TII) or formula (TIII) wherein each R ^a is methyl, u1 is 3-5, u2 is 0-3, and u3 and u4 are each independently 1-4.

In some embodiments, the head group has a structure of one of the following formulas

i);

ii)Wherein each R ₂₀ and R ₃₀ is independently C ₁-C₃ alkyl.

iii)Wherein:

W is Wherein:

Each R ₆、R₇ and R ₈ is independently H or methyl, and

Each of u and t is independently 1, 2 or 3, or

W isWherein:

R ₁₆ is H or = O;

R ₁₄ is a nitrogen-containing 5-or 6-membered heterocycle, NR ₁₀R₁₁、C(O)NR₁₀R₁₁、NR₁₀C(O)NR₁₀R₁₁ or NR ₁₀C(S)NR₁₀R₁₁, wherein each R ₁₀ and R ₁₁ is independently H or C ₁-C₃ alkyl, and

Each of u and v is independently 1, 2 or 3, or

W isWherein:

each R ₆ is independently H or methyl;

each u is independently 1,2 or 3, and

V is C ₂-C₆ alkylene or C ₂-C₆ alkenylene, or

W isWherein:

each R ₆ is independently H or methyl;

Each R ₇ is independently H;

Each R ₈ is methyl;

each u is independently 1,2 or 3, and

V is C ₂-C₆ alkylene or C ₂-C₆ alkenylene, or

W isWherein:

each u is independently 1,2 or 3, and

T is a divalent nitrogen-containing 5-or 6-membered heterocycle, or

W is

Wherein:

each u is independently 1,2 or 3;

Q is O;

Each Z is independently NR ₁₂, and

R ₁₂ is H or C ₁-C₃ alkyl, and

iv)Or (b)Wherein:

W is hydroxy, substituted or unsubstituted hydroxyalkyl, one of the following moieties:

And Wherein

Each Q is independently absent 、-O-、-C(O)-、-C(S)-、-C(O)O-、-(CH₂)_q-C(R⁷)₂-、-C(O)N(R⁷)-、-C(S)N(R⁷)- or-N (R ⁷);

R ⁶ is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N (R ⁷) 2, amino, alkylamino, aminoalkyl, thiol alkyl or N ⁺(R⁷)₃ -alkylene-Q-;

Each R ⁸ is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol alkyl, heterocyclyl, heteroaryl, or two R ⁸ together with the nitrogen atom form a ring, optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino or aminoalkyl groups;

q is 0, 1,2, 3, 4 or 5, and

P is 0, 1,2, 3, 4 or 5.

In some embodiments, the ionizable lipid is a compound of table I, table II, table III, or table IV.

In some embodiments, the ionizable lipid is a 2272 or 2243 lipid.

In the CLP or LNMP formulation or the lipid nanoparticle composition, more than one ionizable lipid may be used for the ionizable lipid component, one or more of the ionizable lipids of the compounds of formula from groups i) -iv) may be used alone or in combination with a different ionizable lipid of the compounds of formula from groups i) -iv).

In some embodiments, the polynucleotide is a polynucleotide construct that encodes one or more wild-type or engineered antigens (or antibodies to antigens). The antigen may be derived from a tumor, such as a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof. In some embodiments, the polynucleotide construct encodes anti-TNF.

In some embodiments, the antigenic polypeptide encoded by the polynucleotide is a tumor antigen polypeptide, including p53, ART-4, BAGE, ss-catenin /m、Bcr-abL CAMEL、CAP-1、CASP-8、CDC27/m、CDK4/m、CEA、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 (e.g., 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, MC R, myosin/m, MUC1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl 90-BCR abL (pl 90 minor BCR-abL), plac-1, l/RAE 3, rt-3, SCP-3, or SCP-1, TRP 2, or a combination thereof, or a combination of three or two or three thereof.

In some embodiments, the antigenic polypeptide encoded by the polynucleotide is a tumor antigen polypeptide, including CD2、CD3、CD4、CD8、CD11b、CD14、CD16、CD19、CD20、CD22、CD25、CD27、CD33、CD37、CD38、CD40、CD44、CD45、CD47、CD52、CD56、CD70、CD79、CD137、4-IBB、5T4、AGS-5、AGS-16、 angiogenin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, cripto, ED-B, erbBl, erbB2, erbB3, erbB4, EGFL7, epCAM, ephA2, ephA3, ephB2, FAP, fibronectin, folate receptor, foxp3, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplo, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF R, integrin av, integrin ανβ, LAG-3, lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, fibronectin-4, KGD2, ch, OX40, TIM 1-72, anti-5, t1, t2, and variants thereof, and the like, as well as variants of VEGFR 1, VEGFR 2, and the like.

In some embodiments, the tumor antigen polypeptide is an IL2 peptide, IL-2-Ra, anti-CD 19 antibody, anti-CD 20 antibody, chimeric antigen receptor T cell (CAR-T) antibody, anti-HER 2 antibody, etanercept (e.g., enbrel), adalimumab (e.g., humira), epoetin alfa (e.g., epogen), feglastin (filgrastim) (e.g., neupogen), pembrolizumab (e.g., kerpareizumab (Keytruda), rituximab (rituximab) (e.g., rituximab), romidepsin (romiplostim) (e.g., nplate), saxadine (sargramostim) (e.g., leukine), or a fragment or subunit thereof. In one embodiment, the tumor antigen polypeptide is an IL2 peptide or a fragment or subunit thereof. In one embodiment, the tumor antigen polypeptide is alfazoptin (epoetin alfa) (e.g., epogen) or a fragment or subunit thereof.

In some embodiments, the tumor antigen polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof. In one embodiment, the tumor antigen polypeptide comprises a lung cancer antigen.

In some embodiments, the polynucleotide may be mRNA, siRNA or siRNA precursor, microRNA (miRNA) or miRNA precursor, plasmid, dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), peptide Nucleic Acid (PNA), morpholino, locked Nucleic Acid (LNA), piwi interacting RNA (piRNA), ribozyme, deoxyribose enzyme (DNAzyme), aptamer, circular RNA (circRNA), guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is mRNA. In one embodiment, the polynucleotide is circRNA.

In one embodiment, the polynucleotide is an mRNA encoding a molecule comprising an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence provided in any one of table 3.

In one embodiment, the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of an IL-2 molecule provided in any of tables V-VII and 3.

In some embodiments, the mRNA is (a) a DNA molecule or (b) an RNA molecule. In the mRNA, T is optionally replaced by U.

In some embodiments, the mRNA is a DNA molecule. The DNA molecule may also comprise a promoter. In some embodiments, the promoter is a T7 promoter, a T3 promoter, or an SP6 promoter. In some embodiments, the promoter is located in the 5' UTR.

In some embodiments, the mRNA is an RNA molecule. The RNA molecule may be a self-replicating RNA molecule.

In some embodiments, the mRNA is an RNA molecule. The RNA molecule may further comprise a 5' cap. The 5' Cap may have a Cap 1 structure, a Cap 1 (m 6A) structure, a Cap 2 structure, a Cap 3 structure, a Cap 0 structure, or any combination thereof.

In some embodiments, the polynucleotide is an mRNA encoding an IL-2 molecule. In one embodiment, the IL-2 molecule includes a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof. In one embodiment, the IL-2 molecule includes a naturally occurring variant of an IL-2 molecule (e.g., an IL-2 variant, such as described herein), or a fragment thereof.

In some embodiments, the mRNA comprises a5 'untranslated region (UTR) and/or a 3' UTR.

In some embodiments, the mRNA comprises a 5' UTR. The 5' UTR may comprise a Kozak sequence.

In some embodiments, the mRNA comprises a 3' UTR. In some embodiments, the 3' UTR comprises one or more sequences derived from an amino terminal cleavage enhancer (amino-TERMINAL ENHANCER of split, AES). In some embodiments, the 3' UTR comprises a sequence derived from a mitochondrially encoded 12S mRNA (mtRNRl).

In some embodiments, the mRNA comprises a poly (a) sequence. In one embodiment, the poly (a) sequence is a 110 nucleotide sequence consisting of a sequence of 30 adenosine residues, a 10 nucleotide linker sequence, and a sequence of 70 adenosine residues.

In some embodiments, the natural lipids of LNMP are extracted from a plant source such as lemon or algae. In some embodiments, the natural lipid is extracted from lemon. In some embodiments, the natural lipid is extracted from a bacterial source such as e.coli (e.coli) or salmonella typhimurium (Salmonella typhimurium).

In some embodiments, for the ionizable lipid composition, the ionizable lipid of the compound of formula from groups i) -iv) may be used in combination with one or more other ionizable lipids. For example, one or more other ionizable lipids may include 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5, SM-102 (lipid H), and ALC-315. In one embodiment, the additional ionizable lipid is comprised between C12 and 200.

In some embodiments, the LNMP is produced by a method comprising lipid extrusion. In some embodiments, the LNMP is produced by a method comprising treating a solution comprising a lipid extract of PMP in a microfluidic device comprising an aqueous phase, thereby producing the LNMP. In some embodiments, the aqueous phase comprises the polynucleotide. In some embodiments, the reconstitution is performed in the presence of sterols, thereby producing LNMP comprising natural lipids, ionizable lipids, and sterols. In some embodiments, the sterol is cholesterol or sitosterol.

In some embodiments, the reconstitution is performed in the presence of a pegylated lipid (or PEG-lipid conjugate), thereby producing LNMP comprising a natural lipid, the ionizable lipid, and PEG-lipid conjugate. In some embodiments, the LNMP further comprises a sterol and polyethylene glycol (PEG) -lipid conjugate.

In some embodiments, the LNP composition comprises one or more ionizable lipids, one or more synthetic structural lipids, sterols, and one or more PEG-modified lipids.

In some embodiments, the LNMP or the LNP composition further comprises a sterol and polyethylene glycol (PEG) -lipid conjugate.

In some embodiments, the PEG-lipid conjugate is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some embodiments, the PEG-lipid conjugate is PEG-DMG or PEG-PE. In some embodiments, the PEG-DMG is PEG2000-DMG or PEG2000-PE.

In some embodiments, the synthetic structural lipid of the LNP composition is a phospholipid selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dimetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (POPC), dioleoyl phosphatidylethanolamine (POPE), palmitoyl Oleoyl Phosphatidylethanolamine (POPG), dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), mono-phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (DSPE), stearoyl phosphatidylethanolamine (DSPE), phosphatidylethanolamine (stearoyl phosphatidylethanolamine (DEPE), and mixtures thereof.

In some embodiments, the LNP composition comprises:

about 20 mole% to about 50 mole% of said ionizable lipid,

From about 5 mole% to about 60 mole% of the synthetic structural lipid,

About 7 mole% to about 50 mole% of said sterols, and

About 0.5 mole% to about 3 mole% of the polyethylene glycol (PEG) -lipid conjugate.

In some embodiments, the LNP composition comprises:

about 20 mole% to about 50 mole% of an ionizable lipid of Table I, table II, table III, or Table IV,

From about 5 mole% to about 60 mole% of the synthetic structural lipid,

About 7 mole% to about 50 mole% of said sterols, and

In some embodiments, the LNP composition comprises an ionizable lipid to synthetic structural lipid to sterol of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:30:32.5:2.5, about 35:16:46.5:2.5, about 35:25:37.5:2.5, about 35:40:22.5:2.5, about 45:10:43.5:1.5, about 50:20:28.5:1.5, or about 50:10:38.5:1.5.

In some embodiments, the LNMP comprises:

about 20 mole% to about 50 mole% of said ionizable lipid,

From about 5 mole% to about 60 mole% of said natural lipid, optionally neutral lipid,

About 7 mole% to about 50 mole% of said sterols, and

In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:50:12.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:20:42.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:30:32.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:16:46.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:25:37.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:40:22.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 45:10:43.5:1.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 35:16:46.5:2.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 50:20:28.5:1.5. In one embodiment, the LNMP comprises a molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid of about 50:10:38.5:1.5.

In some embodiments, the LNMP can further comprise neutral lipids as helper lipids. In some embodiments, the natural lipids may be used in combination with neutral lipids as structural lipid components. Neutral lipids can be used in a molar ratio of neutral lipids to natural lipids of 10:1 to 1:10 or 3:1 to 1:3 (e.g., 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10). Non-limiting examples of neutral lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), palmitoyl phosphatidylethanolamine (POPE), palmitoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (spp), stearoyl phosphatidylethanolamine (DEPE), and mixtures thereof. In one embodiment, the structural lipid component in the LNMP may comprise natural lipid+dope, or natural lipid+dspc.

Thus, in these embodiments, the LNMP comprises an ionizable lipid as described herein, a structural lipid comprising a natural lipid and a neutral lipid, a sterol, and/or a PEG-lipid.

In some embodiments, the LNMP comprises:

about 20 mole% to about 50 mole% of said ionizable lipid,

About 5 mole% to about 60 mole% of a structural lipid component (i.e., the natural lipid and the neutral lipid),

About 7 mole% to about 50 mole% of said sterols, and

In one embodiment, the LNMP comprises ionizable lipids (natural lipid + neutral lipid): sterols: PEG-lipids in a molar ratio of about 35:50:12.5:2.5. In one embodiment, the LNMP comprises ionizable lipids (natural lipid + neutral lipid): sterols: PEG-lipids in a molar ratio of about 35:20:42.5:2.5. For example, the LNMP may comprise ionizable lipids (natural lipids+neutral lipids) sterols: PEG-lipids in a molar ratio of about 35 (10+10): 42.5:2.5. In one embodiment, the LNMP comprises ionizable lipids (natural lipid + neutral lipid): sterols: PEG-lipids in a molar ratio of about 50:20:28.5:1.5. For example, the LNMP may comprise ionizable lipids (natural lipids+neutral lipids) sterols: PEG-lipids in a molar ratio of about 50 (10+10): 28.5:1.5.

In some embodiments, the LNMP comprises:

Natural lipids extracted from lemon or algae, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG。

In some embodiments, the LNMP comprises:

Natural lipids extracted from lemon or algae, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMPE-PEG2k。

In one embodiment, the LNMP comprises:

natural lipids extracted from lemon, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG or DMPE-PEG2k. The LNMP can comprise ionizable lipids to lemon lipids to cholesterol to DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (lemon lipid + neutral lipid): cholesterol: DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids to lemon lipids to cholesterol to DMG-PEG in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (lemon lipid + neutral lipid): cholesterol: DMG-PEG in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In one embodiment, the LNMP comprises:

Natural lipids, and optionally neutral lipids, extracted from algae,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMPE-PEG2k. The LNMP can comprise ionizable lipids to algae lipids to cholesterol to DMPE-PEG2k in a molar ratio of about 35:20:42.5:2.5, about 35:20:42.5:2, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (algae lipids+neutral lipids): cholesterol: DMPE-PEG2k in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In one embodiment, the LNMP comprises:

Natural lipids, and optionally neutral lipids, extracted from algae,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG. The LNMP can comprise ionizable lipids to algae lipids to cholesterol to DMG-PEG in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (algae lipids+neutral lipids): cholesterol: DMG-PEG in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In some embodiments, the LNMP comprises:

natural lipids extracted from E.coli or Salmonella typhimurium, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMPE-PEG2k。

In one embodiment, the LNMP comprises:

Natural lipids extracted from E.coli, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMPE-PEG2k. The LNMP can comprise ionizable lipids to E.coli lipids to cholesterol to DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (E.coli lipids+neutral lipids): cholesterol: DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In one embodiment, the LNMP comprises:

natural lipids, and optionally neutral lipids, extracted from Salmonella typhimurium,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMPE-PEG2k. The LNMP can comprise ionizable lipids, salmonella typhimurium lipids, cholesterol, DMPE-PEG2k in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (Salmonella typhimurium lipid + neutral lipid): cholesterol: DMPE-PEG2k in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In some embodiments, the LNMP comprises:

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG。

In one embodiment, the LNMP comprises:

Natural lipids extracted from E.coli, and optionally neutral lipids,

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG. The LNMP can comprise ionizable lipids to E.coli lipids to cholesterol to DMG-PEG in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (E.coli lipids+neutral lipids): cholesterol: DMG-PEG in a molar ratio that can be about 35:50:12.5:2.5, about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In one embodiment, the LNMP comprises:

Ionizable lipids from Table I, table II, table III, or Table IV,

Cholesterol, and

DMG-PEG. The LNMP can comprise ionizable lipids to Salmonella typhimurium lipids to cholesterol to DMG-PEG in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5. The LNMP can comprise ionizable lipids (Salmonella typhimurium lipid + neutral lipid): cholesterol: DMG-PEG in a molar ratio of about 35:20:42.5:2.5, or about 50:20:28.5:1.5.

In some embodiments, the LNMP is a lipophilic moiety selected from the group consisting of a lipid complex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, and an emulsion. In one embodiment, the LNMP is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In one embodiment, the LNMP is a lipid nanoparticle.

In some embodiments, the LNMP is less than about 200 nm a in size. In one embodiment, the LNMP is less than about 150 nm in size. In one embodiment, the LNMP is less than about 100 nm in size. In one embodiment, the LNMP is from about 80 nm to about 100 nm in size. In one embodiment, the LNMP is from about 55 nm to about 80 nm in size.

In some embodiments, the LNMP has an N: P ratio of at least 3, e.g., an N: P ratio of 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 6 to 30, 6 to 20, 6 to 15, or 6 to 12.

In some embodiments, the total lipid to polynucleotide weight ratio of the RNA composition is about 50:1 to about 10:1. In one embodiment, the total lipid to polynucleotide weight ratio of the RNA composition is about 44:1 to about 24:1. In one embodiment, the total lipid to polynucleotide weight ratio of the RNA composition is about 40:1 to about 28:1. In one embodiment, the total lipid to polynucleotide weight ratio of the RNA composition is about 38:1 to about 30:1. In one embodiment, the total lipid to polynucleotide weight ratio of the RNA composition is about 37:1 to about 33:1.

In some embodiments, the RNA composition, e.g., aqueous phase, further comprises HEPES or TRIS buffer. The HEPES or TRIS buffer can have a pH of about 7.0 to about 8.5. The HEPES or TRIS buffer can have a concentration of about 7 mg/mL to about 15 mg/mL. The aqueous phase may also contain about 2.0 mg/mL to about 4.0 mg/mL NaCl.

In some embodiments, the RNA composition, e.g., aqueous phase, comprises water, PBS, or citrate buffer. In one embodiment, the aqueous phase comprises a citrate buffer having a pH of about 3.2.

In some embodiments, the aqueous phase and the lipid solution are mixed in a 3:1 volume ratio.

In some embodiments, the RNA composition further comprises one or more cryoprotectants. The one or more cryoprotectants may be sucrose, glycerol, or a combination thereof. In one embodiment, the RNA composition comprises a combination of sucrose at a concentration of about 70 mg/mL to about 110 mg/mL and glycerol at a concentration of about 50 mg/mL to about 70 mg/mL.

In some embodiments, the RNA composition is a lyophilized composition. The lyophilized RNA composition can comprise one or more lyoprotectants. The lyophilized RNA composition can comprise poloxamer (poloxamer), potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized RNA composition comprises a poloxamer (poloxamer), such as poloxamer 188.

In some embodiments, the RNA composition is a lyophilized composition. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1.0% w/w of the polynucleotide. In one embodiment, the lyophilized RNA composition comprises about 1.0 to about 5.0% w/w lipid. In one embodiment, the lyophilized RNA composition comprises about 0.5 to about 2.5% w/w TRIS buffer. In one embodiment, the lyophilized RNA composition comprises about 0.75 to about 2.75% w/w NaCl. In one embodiment, the lyophilized RNA composition comprises about 85 to about 95% w/w sugar, such as sucrose. In one embodiment, the lyophilized RNA composition comprises about 0.01 to about 1.0% w/w poloxamer (poloxamer), such as poloxamer 188. In one embodiment, the lyophilized RNA composition comprises about 1.0 to about 5.0% w/w potassium sorbate.

In another aspect, provided herein is a method of delivering an RNA composition in a subject, comprising administering the RNA composition discussed in the above aspects of the invention to the subject.

In another aspect, provided herein is a method of inducing an immune response in a subject, comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

In another aspect, provided herein is a method of treating or preventing cancer in a subject comprising administering to the subject the RNA composition discussed in the above aspects of the invention.

In some embodiments, the LNMP is a lipophilic moiety selected from the group consisting of a lipid complex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a platelet, a micelle, and an emulsion. In one embodiment, the LNMP is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes. In one embodiment, the LNMP is a lipid nanoparticle.

In some embodiments, the LNMP is less than about 200 nm a in size. In one embodiment, the LNMP is less than about 150 nm in size. In one embodiment, the LNMP is less than about 100 nm in size. In one embodiment, the LNMP is from about 55 nm to about 80 nm in size.

In some embodiments, the methods provided herein include lipid nanoparticles having a bacterially derived lipid composition comprising (a) a natural component comprising one or more lipids extracted from a natural source, and (b) an ionizable lipid.

In some embodiments, the natural source is selected from bacteria, animals, insects, archaea, and fungi.

In some embodiments, the methods provided herein include lipid nanoparticles having a bacterially derived lipid composition comprising (a) a bacterial component comprising one or more lipids extracted from a bacterial source, and (b) an ionizable lipid.

One or more lipids extracted from a bacterial source, and (b) ionizable lipids to target cells.

In some embodiments, the lipid nanoparticle comprises purified bacterial lipid of bacterial component (a) in the presence of ionizable lipid (b) to produce a bacterial-derived lipid composition.

In some embodiments, the bacterial source is selected from the group consisting of Escherichia (Escherichia), acinetobacter (Acinetobacter), agrobacterium (Agrobacterium), anabaena (Anabaena), aquifex (Aquifex), azotobacter (Azoarcus), azotobacter (Azotobacter), bao Te (Bordetella), rhizobium (Bradyrhizobium), brucella (Brucella), brucella (Buchnera), bruchnera (Buchnera), Burkholderia (Burkholderia), candidatus, chromobacterium (Chromobacterium), chlorella (Crocosphaera), dechloromonas (Dechloromonas), desulfurous bacteria (Desulfitobacterium), de-sulfation small bacillus (Desulfotalea), erwinia (Erwinia), francisella (FRANCISELLA), fusobacterium (Fusobacterium), myxobacterium (Gloeobacter), Gluconobacter (Gluconobacter), helicobacter (Helicobacter), legionella (Legionella), magnetic spirochete (Magnetospirillum), mesorhizobium (Mesorhizobium), methylococcus (Methylococcus), neisseria (Neisseria), nitrosomonas (Nitrosomonas), nostoc (Nostoc), photobacterium (Photobacterium), photorhabdus (Photorhabdus), neisseria (Photorhabdus), The species of genus Geomonas (Polaromonas), genus Protococcus (Prochlorococcus), genus Pseudomonas (Pseudomonas), genus Acidophilia (Psychrobacter), genus Ralstonia (Ralstonia), genus Rhodotorula (Rubrivivax), genus Salmonella (Salmonella), genus Shewanella (Shewanella), genus Shigella (Shigella), genus Sinorhizobium (Sinorhizobium), genus Synechococcus (Synechococcus), Synechocystis (Synechocystis), thermosynechococcus (Thermotoga), thermotoga (Thermotoga), thermus (Thermus), thiobacillus (Thermobacillus), shu Maozao (Trichodesmium), vibrio (Vibrio), weiger Wo Sijun (Wigglesworthia), wo Linshi (Wolinella), xanthomonas (Xanthomonas), trichoderma (Xylella), Yersinia pestis (Yersinia), bacillus (Bacillus), clostridium (Clostridium), deinococcus (Microbacterium), geobacillus (Exiguobacterium), lactobacillus (Geobacillus), lactobacillus (Lactobacillus), morchella (Moorella), bacillus (Oceanobacillus), symbiotic Bacillus (Symbiobacterium) and Thermoanaerobacter (Thermoanaerobacter). In one embodiment, the bacterial source is Escherichia (e.g., E.coli). In one embodiment, the bacterial source is Salmonella (Salmonella) (e.g., salmonella typhimurium (Salmonella typhimurium)).

In some embodiments, the bacterial component comprises isolated bacterial extracellular vesicles.

In some embodiments, the bacterial component is modified by reconstituting a membrane comprising the bacterial component in the presence of the ionizable lipid.

In some embodiments, the bacterial component is modified by reconstituting a membrane of purified bacterial lipid comprising the bacterial component with the ionizable lipid.

In some embodiments, the ionizable lipid has one or more characteristics selected from the group consisting of:

(i) At least 2 ionizable amines;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

The invention provides LNP compositions or LNMPs comprising one or more active agents or therapeutic agents, methods of making lipid particles, and methods of delivering and/or administering lipid particles (e.g., for treating a disease or disorder).

In a preferred embodiment, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent or therapeutic agent in the lipid particle is resistant to enzymatic degradation (e.g., by nucleases or proteases) in aqueous solution. In other preferred embodiments, the lipid particle is substantially non-toxic to mammals such as humans.

In one aspect, provided herein is a particle comprising:

(a) A nucleic acid;

(b) An ionizable lipid comprising less than 50 mole% of the total lipids in said particles;

(c) A composition of natural source lipids comprising 10 to 90 mole% of the total lipids in the particles;

(d) Sterols (e.g., cholesterol or derivatives thereof) that make up 5 to 40 mole% of the total lipids in the particles, and

(E) Conjugated lipids (e.g., PEG-lipids) that inhibit aggregation of particles, which constitute 0.5 to 3 mole% of the total lipids in the particles.

In another aspect, provided herein is a particle comprising:

(a) A nucleic acid;

(b) An ionizable lipid comprising from about 10 mole% to about 45 mole% of the total lipids in said particles;

(c) A composition of natural source lipids comprising from about 10 mole% to about 90 mole% of the total lipids in the particles;

(d) Sterols (e.g., cholesterol or derivatives thereof) comprising from about 10 mole% to about 50 mole% of the total lipids in the particles, and

(E) Conjugated lipids (e.g., PEG-lipids) that inhibit aggregation of particles, which constitute from 0 mole% to about 10 mole% of the total lipids in the particles.

In certain embodiments, the LNP or LNMP particle comprises (a) a nucleic acid (e.g., mRNA or circRNA), (b) an ionizable lipid that comprises from about 20 mole% to about 45 mole% of the total lipids in the particle, (c) a non-ionizable lipid that comprises from about 13 mole% to about 49.5 mole% of the total lipids in the particle, and (d) a conjugated lipid that inhibits aggregation of particles that comprises from about 0.5 mole% to about 2 mole% of the total lipids in the particle.

In some embodiments, the LNP or LNMP particle comprises (a) mRNA or circRNA, (b) an ionizable lipid that comprises from about 25 mole% to about 35 mole% of the total lipid in the particle, (c) cholesterol or a derivative thereof that comprises from about 31.5 mole% to about 42.5 mole% of the total lipid in the particle, and (d) a PEG-lipid conjugate that comprises from about 1 mole% to about 2 mole% of the total lipid in the particle.

In some embodiments, the LNP or LNMP particle comprises (a) mRNA or circRNA, (b) an ionizable lipid that comprises from about 52 mole% to about 62 mole% of the total lipid in the particle, (c) a mixture of phospholipid and cholesterol or derivatives thereof that comprises from about 36 mole% to about 47 mole% of the total lipid in the particle, and (d) a PEG-lipid conjugate that comprises from about 1 mole% to about 2 mole% of the total lipid in the particle.

In some embodiments, provided herein are methods for treating a disease or disorder in a mammalian subject in need thereof, the methods comprising orally administering to the mammalian subject a therapeutically effective amount of LNP or LNMP described herein.

In some embodiments, provided herein are compositions in the form of enteric coated capsules, tablets, caplets, or multiparticulate carriers (e.g., granules, microparticles, pellets, and beads).

In some embodiments, provided herein is a method and delivery system for administering a polynucleotide, wherein a drug, bile salt or bile acid, and at least one surfactant are present in a single dosage form.

In some embodiments, provided herein is a dosage form comprising an osmotically activated device, wherein the semipermeable membrane encapsulates a bile salt or bile acid, at least one surfactant provided herein, and a hydrophilic drug.

In some embodiments, provided herein is a delayed release pharmaceutical dosage form for oral administration of low molecular weight heparin, wherein the dosage form comprises a composition of (a) a therapeutically effective amount of low molecular weight heparin, (b) bile salts or bile acids, (c) at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof, and (d) means for delayed release of the composition from the dosage form after oral administration. In a preferred embodiment, the composition further comprises a solubilizing agent to ensure good solubilization and/or dissolution of one or more components in the composition.

In some embodiments, the size, shape, or general configuration of the dosage form is not limited and may include, for example, a capsule, tablet, or caplet, or a plurality of granules, microparticles, beads, or pellets, which may or may not be encapsulated. Furthermore, heparin or bile salts or cholic acid may be present as a coating. In addition, the dosage form or components of the dosage form may be enteric coated, for example, capsules or tablets may be enteric coated, multiparticulate dosage forms such as drug-containing particles, pellets, microparticles and beads may also be enteric coated. The enteric coating will typically comprise bioerodible, gradually hydrolyzable and/or gradually water soluble materials suitable to provide the desired delayed release profile.

In some embodiments, any bile salts or bile acids may be used, provided that the selected compound is at least partially dissolved or suspended in the composition.

To ensure good solubilization and/or dissolution of bile salts or bile acids and to minimize precipitation thereof, additional formulation auxiliary excipients may be added to the above dosage form. Such excipients include, for example, buffers, co-solvents, complexing agents, and crystal growth inhibitors. In addition, processing techniques such as size reduction, co-precipitation, coagulation, lyophilization, spray drying, eutectic mixing, solid solution, or other suitable techniques may be used to make bile salts or bile acids more readily and rapidly soluble. If suspended, the bile salts or bile acids may be in any of a variety of forms, for example crystalline, amorphous, nanosized, micronized or milled.

In some embodiments, suitable hydrophilic surfactants typically have an HLB value of at least 10, while suitable lipophilic surfactants typically have an HLB value of about 10 or less than about 10. Co-administration of low molecular weight heparin with bile salts or bile acids and at least one surfactant provided herein significantly enhances transmembrane absorption of the drug.

While not wishing to be bound by theory, it is proposed herein that a substantially homogeneous optically clear aqueous dispersion produced immediately upon contact with an aqueous medium, such as gastrointestinal fluids, makes the drug immediately available for bioabsorption, i.e., the drug is rapidly and effectively "presented" to a target absorption site in the body. The resulting optically clear aqueous dispersion is generally characterized by an absorbance at 400 nm of less than about 0.3 measured at 100X dilution. In another embodiment, a method for administering low molecular weight heparin to a patient is provided, the method comprising administering a therapeutically effective amount of the polynucleotide with bile salts or bile acids and at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. Typical dosages of low molecular weight heparin for oral administration using the dosage form of the present invention are about 700 to 400,000 IU/day, typically ranging from about 2,500 to 10,000 IU/day, whereas typical dosages of common (unfractionated) heparin for oral administration are about 2,500 to 800,000 units/day. Typically, the medicament is administered for the treatment or prevention of thrombosis.

In some embodiments, a drug delivery system is provided comprising an osmotically activated device (i.e., osmotically activated tablet or capsule) that contains a therapeutically effective amount of a hydrophilic drug, bile salt or bile acid, and at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. In this embodiment, the drug-containing composition is encapsulated in a semipermeable membrane or barrier that includes pores. As is known in the art with respect to so-called "osmotic pump" drug delivery devices, the semipermeable membrane allows the passage of water in either direction, but does not allow the passage of drugs or other components of the drug-containing composition. Thus, when the device is exposed to an aqueous liquid, water will flow into the device due to the osmotic pressure difference between the inside and the outside of the device, and when water flows into the device, the internal drug-containing formulation will be "pumped" out through the pores. The drug release rate dD/dt is equal to the inflow rate of water multiplied by the drug concentration. In a preferred embodiment, the osmotic activation device is enteric coated with a coating material effective to provide a desired delayed release profile.

In some embodiments, a drug delivery system for oral administration of a polysaccharide drug is provided, the system consisting of a first dosage form and a second dosage form, wherein the first dosage form comprises a therapeutically effective amount of the polysaccharide drug and the second dosage form comprises a bile salt or bile acid in combination with at least one surfactant selected from the group consisting of hydrophilic surfactants, lipophilic surfactants and mixtures thereof, wherein at least one dosage form is a delayed release dosage form, e.g. coated with an enteric coating. The polysaccharide drug may be, for example, glucosamine, glycosaminoglycans, dextran, xylan, pentasaccharide, polygalacturonic acid, polymannuronate, chitin, pharmaceutically acceptable salts, esters, or other derivatives thereof, and any combination of the foregoing. The dosage forms may be administered simultaneously or sequentially, in which case the first dosage form may be administered first followed by the second dosage form, or the second dosage form may be administered first followed by the first dosage form.

In some embodiments, the RNA composition, e.g., aqueous phase, further comprises HEPES or TRIS buffer. The pH of HEPES or TRIS buffer can be about 7.0 to about 8.5. The HEPES or TRIS buffer can have a concentration of about 7 mg/mL to about 15 mg/mL. The aqueous phase may also contain about 2.0 mg/mL to about 4.0 mg/mL NaCl.

In some embodiments, the RNA composition is a lyophilized composition. The lyophilized mRNA composition may comprise one or more lyoprotectants. The lyophilized RNA composition can comprise poloxamer (poloxamer), potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized RNA composition comprises a poloxamer (poloxamer), such as poloxamer 188.

In another aspect, provided herein is a method of delivering RNA in a subject comprising administering to a subject the RNA composition discussed in the above aspects of the invention.

In these aspects of the invention, the RNA composition may be administered orally, intravenously, intradermally, intramuscularly, intranasally, intraocularly or rectally and/or subcutaneously. In certain embodiments, the RNA composition is administered orally, enterally, intravenously, intramuscularly, and/or subcutaneously.

In some embodiments, the RNA composition is administered at a dosage level sufficient to deliver from about 0.01 mg/kg to about 0.2 mg/kg of RNA to the subject. In some embodiments, the RNA composition is administered at a dosage level sufficient to deliver 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg of RNA to the subject.

In some embodiments, the RNA composition is administered to the subject one, two, three, four or more times. In some embodiments, the RNA composition is administered to the subject one or two times.

In some embodiments, the method further comprises administering an additional therapeutic agent to the subject.

In some embodiments, the additional therapeutic agent is an anti-cancer therapeutic agent.

In some embodiments, the additional therapeutic agent is a therapeutic agent that treats and/or prevents chronic pain. In one embodiment, the additional therapeutic agent is an opioid analgesic, such as buprenorphine (buprenorphine), a non-steroidal anti-inflammatory drug (NSAID) (such as meloxicam (meloxicam) SR), or a combination thereof.

In some embodiments, the additional therapeutic agent is administered prior to, concurrently with, or after administration of the RNA composition.

Definition of the definition

As used herein, the term "effective amount," effective concentration, "or" concentration effective..once again, refers to an amount of LNMP or nucleic acid composition sufficient to achieve the recited result or to achieve a target level (e.g., a predetermined level or threshold level) within or on a target organism.

As used herein, the term "therapeutic agent" refers to an agent that can act on an animal, such as a mammal (e.g., a human), an animal pathogen, or a pathogen carrier, such as an antifungal agent, an antibacterial agent, a virucide, an antiviral agent, an insecticide, a nematicide, an antiparasitic agent, or an insect repellent. As defined herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to linear or branched single-or double-stranded RNA or DNA or hybrids thereof, regardless of length (e.g., at least 2,3,4, 5,6,7,8, 9,10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also includes RNA/DNA hybrids. The nucleotides in a nucleic acid are typically linked by phosphodiester bonds, but the term "nucleic acid" also includes nucleic acid analogs having other types of bonds or backbones (e.g., bonds or backbones of phosphoramides, phosphorothioates, phosphorodithioates, O-methyl phosphoramidates, morpholinos, locked Nucleic Acids (LNA), glycero Nucleic Acids (GNA), threose Nucleic Acids (TNA), and Peptide Nucleic Acids (PNA), etc.). The nucleic acid may be single stranded, double stranded or comprise part of a single stranded and double stranded sequence. The nucleic acid may comprise any combination of deoxyribonucleotides and ribonucleotides and any combination of bases including, for example, adenine, thymine, cytosine, guanine, uracil and modified or non-classical bases (including, for example, hypoxanthine, xanthine, 7-methylguanine, 5, 6-dihydrouracil, 5-methylcytosine and 5-hydroxymethylcytosine).

As used herein, the terms "circRNA", "cyclic polyribonucleotide", "cyclic RNA" and "cyclic polyribonucleotide molecule" are used interchangeably to refer to polyribonucleotide molecules having a structure that does not have a free end (i.e., no free 3 'end and/or 5' end), such as polyribonucleotide molecules that form a cyclic or non-terminal structure by covalent bonds (e.g., covalent closure) or non-covalent bonds. The cyclic polyribonucleotide may be, for example, a covalently closed polyribonucleotide.

As used herein, the term "expression sequence" is a nucleic acid sequence encoding a product, such as a polypeptide or regulatory nucleic acid. An exemplary expression sequence encoding a polypeptide may comprise a plurality of nucleotide triplets, each of which may encode an amino acid and is referred to as a "codon".

As used herein, the terms "linear RNA", "linear polyribonucleotide" and "linear polyribonucleotide molecule" are used interchangeably to refer to a single or polyribonucleotide molecule having a 5 'end and a 3' end. One or both of the 5 'and 3' ends may be free or have another moiety attached. In some embodiments, the linear RNA has a 5 'end or a 3' end modified or protected from degradation (e.g., by a 5 'end protecting agent or a 3' end protecting agent). In some embodiments, the linear RNA has a non-covalently linked 5 'or 3' end. The linear RNA may be used as starting material for cyclization by, for example, a splint linkage (splint ligation) or chemical, enzymatic, ribozyme or splice-catalyzed cyclization methods.

As used herein, the term "polyribonucleotide cargo" herein includes any sequence comprising at least one polyribonucleotide. In embodiments, the polyribonucleotide cargo comprises one or more expression sequences, wherein each expression sequence encodes a polypeptide. In embodiments, the polyribonucleotide cargo comprises one or more non-coding sequences, such as polyribonucleotides with regulatory or catalytic functions. In embodiments, the polyribonucleotide cargo comprises a combination of an expression sequence and a non-coding sequence. In embodiments, the polyribonucleotide cargo comprises one or more of the polyribonucleotide sequences described herein, such as one or more regulatory elements, internal Ribosome Entry Site (IRES) elements, or spacer sequences.

As used herein, nucleic acid elements are "operably linked" or "operably linked" if they are located on a vector such that they can be transcribed to form a linear RNA, which can then be circularized into a circular RNA using the methods provided herein.

As used herein, a "spacer" or "spacer sequence" refers to any contiguous non-coding nucleotide sequence (e.g., a nucleotide sequence of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. Exemplary spacer sequences include, but are not limited to, poly (X) sequences as described herein, repeated or random non-coding DNA or RNA sequences located 3 'or 5' to the open reading frame, or 3 'or 5' untranslated regions. The present disclosure contemplates any spacer sequence that the skilled artisan deems suitable for the polyribonucleotides described herein.

As used interchangeably herein, the terms "poly (X)" and "poly (X) sequence" refer to an untranslated contiguous region of any nucleic acid molecule that is at least 5 nucleotides in length and consists of individual adenine (a), thymine (T), cytosine (C), guanine (G) or uracil (U) residues, or some combination thereof. For example, in some embodiments, the poly (a) sequence may be an adenine residue sequence. In other embodiments, the poly (A-T) sequence is a combination of adenine and thymine residues. In other embodiments, the poly (A-U) sequence may be a combination of adenine and uracil residues. In some embodiments, the poly (A-G) sequence is a combination of adenine residues and guanine residues. In some embodiments, the poly (G-C) sequence is a combination of guanine residues and cytosine residues. In some embodiments, the poly (X) sequence can be at least about 50 nucleotides to about 700 nucleotides in length, can be at least about 60 nucleotides to about 600 nucleotides in length, can be at least about 70 nucleotides to about 500 nucleotides in length, can be at least about 80 nucleotides to about 400 nucleotides in length, can be at least about 90 nucleotides to about 300 nucleotides in length, and can be at least about 100 nucleotides to about 200 nucleotides in length. In some embodiments, the poly (X) sequence can be at least about 50, at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, or at least about 700 nucleotides in length. In some embodiments, the poly (X) sequence can be located 3 '(e.g., downstream) of an open reading frame (e.g., an open reading frame encoding a polypeptide), and the poly (X) sequence can be located 3' of a termination element (e.g., a stop codon) such that the poly (X) sequence is not translated. In some embodiments, the poly (X) sequence may be located 3 'of the termination element and the 3' spacer sequence.

As used herein, the terms "gapped RNA," "gapped linear polyribonucleotide," and "gapped linear polyribonucleotide molecule" are used interchangeably to refer to polyribonucleotide molecules having a 5 'end and a 3' end resulting from cleavage or degradation of a circular RNA.

As used herein, the term "peptide," "protein" or "polypeptide" includes any chain of naturally or non-naturally occurring amino acids (D-amino acids or L-amino acids), regardless of length (e.g., at least 2、3、4、5、6、7、10、12、14、16、18、20、25、30、40、50、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000 or more than 1000 amino acids), presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation) or presence of, for example, one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide, and includes, for example, natural proteins, synthetic or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics. The size of the polypeptide may be, for example, at least 0.1 kD, at least 1 kD, at least 5 kD, at least 10 kD, at least 15 kD, at least 20 kD, at least 30 kD, at least 40 kD, at least 50 kD, or more than 50 kD. The polypeptide may be a full-length protein. Or the polypeptide may comprise one or more domains of a protein.

As used herein, the term "animal" refers to human and non-human animals (including, for example, dogs, cats, horses, rabbits, zoo animals, cattle, pigs, sheep, chickens, and non-human primates).

As used herein, the term "infection" refers to the presence or colonization of a pathogen in an animal (e.g., in one or more parts of an animal), on an animal (e.g., on one or more parts of an animal), or in a habitat surrounding an animal, particularly where the infection reduces the health of the animal (e.g., by causing a disease, disease symptom, or immune (e.g., inflammatory) response).

As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes a disease or disease symptom in an animal by, for example, (i) directly infecting the animal, (ii) producing an agent (agent) (e.g., a pathogenic toxin producing bacterium, etc.) that causes the disease or disease symptom in the animal, and/or (iii) by eliciting an immune (e.g., inflammatory response) in the animal (e.g., biting an insect (e.g., bed bug). As used herein, pathogens include, but are not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids and viruses or any combination thereof, wherein each pathogen is capable of eliciting a disease or symptom in a human by itself or in conjunction with another pathogen.

As used herein, the term "antibody" includes immunoglobulins (whether naturally occurring or partially or fully synthetically produced) and fragments thereof capable of specifically binding to an antigen. The term also encompasses any protein having a binding domain that is homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or may be partially or fully synthetically produced. An "antibody" further includes polypeptides comprising a region from the framework of an immunoglobulin gene or fragment thereof that specifically binds to an antigen and recognizes the antigen. The term "antibody" is used to mean and include whole antibodies, polyclonal antibodies, monoclonal antibodies, and recombinant antibodies, fragments thereof, and further includes single chain antibodies (nanobodies), humanized antibodies, murine antibodies, chimeric mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotypic antibodies, antibody fragments such as, for example, scFv, (scFv) 2, fab 'and F (ab') 2, F (ab 1) 2, fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. "antibody" further includes bispecific antibodies and multispecific antibodies.

As used herein, the term "heterologous" refers to an agent (e.g., a polypeptide) (1) that is exogenous to a plant (e.g., derived from a source that is not a PMP-producing plant or plant part) (e.g., an agent added to a PMP using the loading methods described herein) or (2) that is endogenous to a PMP-producing plant cell or tissue, but is present in the PMP at a higher concentration than found in nature (e.g., higher than found in naturally occurring plant extracellular vesicles) (e.g., added to the PMP using the loading methods, genetic engineering, and in vitro or in vivo methods described herein).

As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, which is described in Altschul et al, (1990) J.mol. Biol. 215:403-41. Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Center for Biotechnology Information).

As used herein, the term "modified NMP" or "modified LNMP" refers to a composition comprising a plurality of NMP or LNMP comprising one or more heterologous agents (e.g., one or more exogenous lipids, such as ionizable lipids, e.g., NMP or LNMP comprising ionizable lipids and sterols and/or pegylated lipids) capable of increasing cellular uptake (e.g., animal cellular uptake, plant cellular uptake, bacterial cellular uptake, or fungal cellular uptake) of NMP or LNMP or a portion or component thereof relative to unmodified NMP or LNMP, the one or more heterologous agents capable of effecting or increasing delivery of a heterologous functional agent (e.g., an agricultural agent or therapeutic agent) to a cell by NMP or LNMP, and/or the one or more heterologous agents capable of effecting or increasing loading (e.g., loading efficiency or capacity) of a heterologous functional agent (e.g., an agricultural agent or therapeutic agent). NMP or LNMP may be modified in vitro or in vivo.

As used herein, the term "unmodified NMP" or "unmodified LNMP" refers to a composition comprising a plurality of NMP or LNMPs lacking a heterologous cell uptake agent capable of increasing NMP cell uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake).

As used herein, the term "modified PMP" or "modified LPMP" refers to a composition that includes a plurality of PMPs or LPMP, the PMP or LPMP including one or more heterologous agents (e.g., one or more exogenous lipids, such as ionizable lipids, e.g., PMP or LPMP comprising ionizable lipids and sterols and/or pegylated lipids) that are capable of increasing cellular uptake (e.g., animal cellular uptake, plant cellular uptake, bacterial cellular uptake, or fungal cellular uptake) of PMP or LPMP or a portion or component thereof relative to unmodified PMP or LPMP, the one or more heterologous agents being capable of achieving or increasing delivery of a heterologous functional agent (e.g., an agricultural agent or therapeutic agent) to a cell via PMP or LPMP, and/or the one or more heterologous agents being capable of achieving or increasing loading efficiency or loading capacity (e.g., an agricultural agent or therapeutic agent). PMP or LPMP can be modified in vitro or in vivo.

As used herein, the term "unmodified PMP" or "unmodified LNMP" refers to a composition comprising a plurality of PMPs or LPMP lacking a heterologous cell uptake agent capable of increasing PMP cell uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake).

As used herein, the term "cellular uptake" refers to the uptake of NMP or LNMP or a portion or component thereof (e.g., a polynucleotide carried by NMP or LNMP) by a cell, such as an animal cell, plant cell, bacterial cell, or fungal cell. For example, uptake may involve transfer of NMP (e.g., LNMP) or a portion of its components from the extracellular environment of a cell into or across the cell membrane, cell wall, extracellular matrix, or into the intracellular environment. Cellular uptake of NMP (e.g., LNMP) can occur via active or passive cellular mechanisms. Cellular uptake includes aspects in which the entire NMP (e.g., LNMP) is taken up by the cell, e.g., by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of the target cell after endocytosis and endosomal escape. In some embodiments, the modified LNMP (e.g., LNMP comprising an ionizable lipid, such as LNMP comprising an ionizable lipid and a sterol and/or a pegylated lipid) has an increased endosomal escape rate relative to the unmodified LNMP. Cellular uptake also includes aspects in which NMP (e.g., LNMP) fuses with the membrane of the target cell. In some embodiments, the one or more polynucleotides are exposed to the cytoplasm of the target cell after membrane fusion. In some embodiments, the LNMP is more fused (e.g., more fused) to the membrane of the target cell relative to the unmodified LNMP.

As used herein, the term "cell penetrating agent" refers to an agent that alters the properties (e.g., permeability) of the cell wall, extracellular matrix, or cell membrane of a cell (e.g., an animal cell, plant cell, bacterial cell, or fungal cell) in a manner that promotes increased cellular uptake relative to cells not contacted with the agent.

As used herein, the term "plant" refers to whole plants, plant organs, plant tissues, seeds, plant cells, seeds, and progeny thereof. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, fruits, harvests, tumor tissue, sap (e.g., xylem sap and phloem sap), and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, and callus tissue). Plant tissue may be in a plant or in a plant organ, tissue or cell culture. In addition, plants can be genetically engineered to produce heterologous proteins or RNAs.

As used herein, the term "bacteria" refers to whole bacteria or portions of bacteria. Bacteria can be further divided into cocci, bacilli, spiralis, or vibrios, different phylum including but not limited to Proteus (Proteus), thick-walled phylum (Firmicutes), bacteroides phylum (Bacteroids), sphingobacteria phylum (sphingobacteria), flavobacterium phylum (Flavobacteria), fusobacterium phylum (Fusobacteria), spiralis phylum (Spirochaetes), viridis phylum (Chlorobia), cyanobacteria phylum (Cyanobacteria), thermal Microbacterium phylum (Thermomicrobia), kidnerella (Xenobacteria), or Water-producing phylum (Aquificae). Examples of specific bacterial species include staphylococcus aureus (Staphylococcus aureus), escherichia coli (ESCHERICHIA COLI), salmonella typhimurium (Salmonella typhimurium), streptococcus pneumoniae (Streptococcus pneumoniae), and pseudomonas aeruginosa (Pseudomonas aeruginosa). Portions of bacteria include cellular components such as peptidoglycans, outer membranes, inner membranes, cell walls, RNA polymerase, metabolites, polypeptides, proteins, flagella, pili, ribosomes, intermediates, cytoplasms, or chromosomes. The bacteria may be genetically engineered to produce heterologous proteins or RNAs, or the bacteria may be genetically engineered to not produce endogenous proteins or RNAs.

As used herein, the term "arthropod" refers to any animal within the phylum arthropoda, or any animal part, organ, tissue, egg, cell, or offspring thereof. Example animals include insects, arachnids and crustaceans. Arthropod cells include, but are not limited to, cells from eggs, suspension cultures, embryos, tissues, organs, exoskeletons, somites, and appendages. Arthropod moieties include body segments, appendages, exoskeletons, eggs, organs, embryos, and various forms of cells and cultures. The arthropod tissue may be in an arthropod, or in an organ, tissue or cell culture. Arthropods can be genetically engineered to produce heterologous proteins or RNAs. Arthropods can be genetically engineered to not produce endogenous proteins or RNAs.

As used herein, the term "fungus" refers to whole fungi, fungal organs, fungal tissues, spores, fungal cells, and their progeny. Example fungi include yeasts, mushrooms, molds (molds) and mildew (mildew). Fungal cells include, but are not limited to, cells from spores, suspension cultures, mycelia, hyphae, thalli, cell walls, tissues, gametophytes, sporophytes, and organs. The fungal tissue may be in a fungus or in an organ, tissue or cell culture. The fungus may be genetically engineered to produce heterologous proteins or RNAs. The fungus may be genetically engineered to not produce endogenous proteins or RNAs.

As used herein, the term "archaea (Archaea)" refers to whole archaea or parts of archaea. Examples of archaea include europaea (euryarchaeota), spring archaea (crenarchaeota), and protoarchaea (koraarchaeota). Parts of archaea include cellular components such as RNA polymerase, glycerol ether lipids, membranes, cell walls, polypeptides, proteins and metabolites. The archaea may be genetically engineered to produce a heterologous protein or RNA, or the archaea may be genetically engineered to not produce an endogenous protein or RNA.

As used herein, the term "plant extracellular vesicles", "plant EVs" or "EVs" refers to closed lipid bilayer structures that naturally occur in plants. Optionally, the plant EV comprises one or more plant EV markers. As used herein, the term "plant EV marker" refers to a component naturally associated with a plant, such as a plant protein, plant nucleic acid, plant small molecule, plant lipid, or combination thereof, including but not limited to any of the plant EV markers listed in the appendix. In some cases, the plant EV marker is a recognition marker for plant EV, but is not a pesticide. In some cases, the plant EV marker is a recognition marker for a plant EV and is also an insecticide (e.g., associated with or encapsulated by multiple PMPs or LPMP, or not directly associated with or not directly encapsulated by multiple PMPs or LPMP).

As used herein, the term "natural messenger package" or "NMP" refers to a lipid structure (e.g., a lipid bilayer, monolayer, multilamellar structure; e.g., a vesicle lipid structure) derived from (e.g., enriched in, isolated from, or purified from) a natural source or a fragment, portion, or extract thereof having a diameter of about 5-2000 nm a (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50-150 nm, or at least 70-120 nm) that includes lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated therewith and that have been enriched in, isolated, or purified from a natural source, a portion of a natural source, or a cell of a natural source, from which the enrichment or isolation removes one or more contaminants or non-desired components. NMP may be a highly purified preparation of naturally occurring EVs. Preferably, at least 1% of one or more contaminants or undesired components from a source, such as a source cell wall component, pectin, organelles (e.g., mitochondria, plastids, e.g., chloroplasts, whitebodies, or amyloplasts, and nuclei), chromatin (e.g., chromosomes), or molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipid-protein structures), are removed (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%). Preferably, NMP has at least 30% purity (e.g., at least 40% purity, at least 50% purity, at least 60% purity, at least 70% purity, at least 80% purity, at least 90% purity, at least 99% purity, or 100% purity) relative to one or more contaminants or undesired components from a natural source, as measured by weight (w/w), spectral imaging (transmittance%) or conductivity (S/m).

As used herein, the term "plant messenger package" or "PMP" refers to a lipid structure (e.g., a lipid bilayer, monolayer, multilamellar structure; e.g., a vesicle lipid structure) derived (e.g., enriched, isolated or purified from) from a plant source or a segment, portion or extract thereof having a diameter of about 5-2000 nm (e.g., at least 5-1000 nm, at least 5-500 nm, at least 400-500 nm, at least 25-250 nm, at least 50-150 nm, or at least 70-120 nm) that includes lipid or non-lipid components (e.g., peptides, nucleic acids, or small molecules) associated therewith and that have been enriched, isolated or purified from a plant, plant part, or plant cell, the enrichment or isolation removing one or more contaminants or non-desired components from the source plant. PMP may be a highly purified preparation of naturally occurring EVs. Preferably, at least 1% of one or more contaminants or undesired components from a source plant, such as a plant cell wall component, pectin, plant organelles (e.g., mitochondria, plastids, e.g., chloroplasts, whitebodies, or amyloplasts, and nuclei), plant chromatin (e.g., plant chromosomes), or plant molecular aggregates (e.g., protein aggregates, protein-nucleic acid aggregates, lipoprotein aggregates, or lipid-protein structures), is removed (e.g., at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%). Preferably, the PMP is at least 30% pure (e.g., at least 40% pure, at least 50% pure, at least 60% pure, at least 70% pure, at least 80% pure, at least 90% pure, at least 99% pure, or 100% pure) relative to one or more contaminants or undesired components from the source plant as measured by weight (w/w), spectral imaging (transmittance%) or conductivity (S/m).

Lipid reconstituted NMP (LNMP) is used herein. For example, lipid-reconstituted PMP (LPMP) is used herein. The terms "lipid-reconstituted NMP" and "LNMP" refer to NMP derived from lipid structures (e.g., lipid bilayer, monolayer, multilamellar structures; e.g., vesicle lipid structures) derived from (e.g., enriched, isolated, or purified from) natural sources, wherein the lipid structures are disrupted (e.g., disrupted by lipid extraction) and reassembled or reconstituted in a liquid phase (e.g., a liquid phase comprising cargo) using standard methods, e.g., by methods comprising lipid membrane hydration and/or solvent injection, to produce LNMP, as described herein. The method may also include sonication, freeze/thaw treatment, and/or lipid extrusion, if desired, for example to reduce the size of reconstituted NMP. Or the LNMP may be generated using a microfluidic device, such as NanoAssemblr x IGNITE ^TM microfluidic instrument (Precision NanoSystems). When the natural source is a plant source, the terms "lipid reconstituted PMP" and "LPMP" are defined in the same manner as "lipid reconstituted NMP" and "LNMP".

As used herein, the term "pure" refers to a PMP preparation in which at least a portion (e.g., at least 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99% or 100%) of a plant cell wall component, plant organelle (e.g., mitochondria, chloroplasts, and nuclei), or plant molecule aggregate (protein aggregate, protein-nucleic acid aggregate, lipoprotein aggregate, or lipid-protein structure) has been removed relative to an initial sample isolated from a plant or portion thereof.

As used herein, the term "composite lipid particle" refers to a lipid particle having a complexation, characterized by comprising a plurality of lipids, including structural lipids extracted from one or more natural sources (e.g., plants or bacteria) and optionally including at least one exogenous ionizable lipid. The composite lipid particle may comprise from 10% w/w to 99% w/w of structural lipids derived from one or more lipid structures of natural origin, e.g. it may comprise at least 10% w/w, at least 20% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w or about 99% w/w of lipids derived from one or more lipid structures of natural origin. In some cases, the complex lipid particles incorporating the natural lipid extract may also be referred to as Natural Messenger Packages (NMP). For example, complex lipid particles incorporating plant lipid extracts may also be referred to as Plant Messenger Packages (PMPs). In some cases, the complex lipid particle incorporating the natural lipid extract and the at least one exogenous ionizable lipid may also be referred to as a lipid reconstituted natural messenger package (LNMP). For example, a composite lipid particle incorporating a plant lipid extract and at least one exogenous ionizable lipid may also be referred to as a lipid reconstituted plant messenger package (LPMP). Thus, any disclosure described herein that relates to LNMP and the features of LNMP formulations is applicable to CLP and CLP formulations.

The composite lipid particle may comprise 3-1000 lipids extracted from one or more natural (e.g., plant, bacterial) sources. The composite lipid particle may comprise natural (e.g., plant, bacterial) lipids from at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of lipids of natural (e.g., plant, bacterial) origin. The composite lipid particle may comprise all or part of the lipid material present in the lipid structure from a natural (e.g. plant, bacterial) source, e.g. it may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or almost 100% of the lipid material present in the lipid structure from a natural source. The complex lipid particle may comprise all or part of the lipid material present in the lipid structure from a particular natural source. For example, it may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or almost 100% of the lipid material present in the lipid structure from a plant source or from a bacterial source.

The composite lipid particle may comprise reduced or minimized proteinaceous matter that is endogenous to the one or more natural (i.e., plant, bacterial) sources, e.g., it may comprise 0% w/w, less than 1% w/w, less than 5% w/w, less than 10% w/w, less than 15% w/w, less than 20% w/w, less than 30% w/w, less than 40% w/w, or less than 50% w/w proteinaceous matter that is endogenous to the one or more natural (e.g., plant, bacterial) sources. In some cases, the lipid bilayer of the composite lipid particle does not contain a protein.

The complex lipid particles may also comprise synthetic structural lipids, such as neutral lipids, as structural lipid component. The structural lipid component of the composite lipid particle may comprise from 10% w/w to 99% w/w of structural lipids derived from synthetic lipid structures (rather than lipids extracted from natural sources), e.g., it may comprise at least 10% w/w, at least 20% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w, or about 99% w/w of lipids derived from synthetic lipid structures.

The complex lipid particle may also comprise at least two exogenous lipids. The composite lipid particle may comprise at least 1% w/w, at least 2% w/w, at least 5% w/w, at least 10% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, or about 90% w/w of the exogenous lipid. Exemplary exogenous lipids include sterols and PEG-lipid conjugates. The composite lipid particle may be used to encapsulate one or more exogenous nucleic acids or polynucleotides encoding one or more peptides, polypeptides, or proteins to enable delivery of the exogenous nucleic acids or polynucleotides to a target cell or tissue.

As used herein, the term "exogenous lipid" refers to a lipid that is exogenous to a natural source (e.g., plant, bacteria), i.e., the lipid is derived from a source that is not the natural source from which the lipid was extracted (e.g., a lipid added to a complex lipid particle formulation using the methods described herein). The term "exogenous lipid" does not exclude lipids of natural origin (e.g. sterols of plant origin). That is, the exogenous lipid may be a plant-derived lipid (e.g., a plant-derived sterol that is exogenous to the plant source from which the lipid was extracted, e.g., the exogenous lipid may be a plant-derived sterol that is added to the complex lipid particle formulation). As another example, the exogenous lipid may be a lipid of natural origin that is exogenous to the particular natural source from which the lipid was extracted (e.g., a lipid of bacterial origin that is exogenous to the plant source from which the lipid was extracted, or vice versa). The exogenous lipid may be a cell penetrating agent, may be capable of increasing delivery of one or more polynucleotides to the cell via the complex lipid formulation, and/or may be capable of increasing loading (e.g., loading efficiency or loading capacity) of the polynucleotide. In some embodiments, the exogenous lipid may be a stabilizing lipid. In some embodiments, the exogenous lipid may be a structural lipid (e.g., a synthetic structural lipid). Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and pegylated lipids.

As used herein, the term "cationic lipid" refers to a positively charged amphiphilic molecule (e.g., a lipid or lipid (lipidoid)) that comprises a cationic group (e.g., a cationic head group).

As used herein, the term "ionizable lipid" refers to an amphiphilic molecule (e.g., a lipid or lipid, such as a synthetic lipid or lipid) that comprises a group (e.g., a head group) that can ionize, e.g., dissociate, under a given condition (e.g., pH) to produce one or more charged species.

Surprisingly, it has been found that ionizable lipids comprising an alkyl chain having multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly suitable for forming lipid particles with increased film mobility. A variety of ionizable lipids and related analogs suitable for use herein have been described in U.S. patent publication nos. 20060083780 and 20060240554, U.S. patent nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613, and 5,785,992, and PCT publication No. WO 96/10390, the disclosures of which are incorporated herein by reference in their entirety.

In some embodiments, the ionizable lipids are ionizable such that they may exist in a positively charged form upon dissociation of pH. Ionization of the ionizable lipid affects the surface charge of the lipid nanoparticle comprising the ionizable lipid under different pH conditions. The surface charge of lipid nanoparticles, in turn, can affect their plasma protein absorption, blood clearance, and tissue distribution (sample, S.C., et al, adv. Drug Deliv Rev 32:3-17 (1998)), and their ability to form non-bilayer structures that can affect endosomal lysis of intracellular delivery nucleic acids (Hafez, I.M., et al, gene Ther 8:1188-1196 (2001)).

In some embodiments, the ionizable lipid is, for example, a lipid that is generally neutral at physiological pH (e.g., pH about 7), but can carry a net charge(s) at acidic pH or basic pH. In one embodiment, the ionizable lipid is one that is generally neutral at a pH of about 7, but can carry a net charge(s) at an acidic pH. In one embodiment, the ionizable lipid is one that is generally neutral at a pH of about 7, but can carry a net charge(s) at an alkaline pH.

In some embodiments, the ionizable lipids do not include those cationic or anionic lipids that normally carry a net charge(s) at physiological pH (e.g., pH about 7).

As used herein, the term "lipid" refers to a molecule that has one or more characteristics of a lipid.

As used herein, the term "stable LNMP formulation" or "stable CLP formulation" refers to a CLP formulation or LNMP composition that has a characteristic of retaining at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) of an initial CLP or LNMP amount (e.g., CLP or LNMP number per milliliter of solution) over a period of time (e.g., at least 24 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days or at least 90 days), relative to the amount of CLP or LNMP in the CLP formulation or LNMP formulation (e.g., at production or formulation), optionally retaining at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 85%, 90%, 95% or 100%) of an initial CLP or LNMP amount (e.g., CLP or LNMP number per milliliter of solution), optionally retaining at least 24% (e.g., at least 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, or 30 ℃) of at least 20% (e.g., at least 20%, 21%, 22%, 23%, 25%, 80%, 15%, or at least 50% (e.g., at least 10-80%) of an activity (e.g., at least 10%) (e.g., at least 10%) of 5-0%) (e.g., 15%) of 5-7-15%) (at or at-0), at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of its activity (e.g., cell wall penetrating activity and/or activity of RNA formulated within CLP or LNMP), optionally at a defined temperature range (e.g., a temperature of at least 24 ℃ (e.g., at least 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, or 30 ℃), at least 20 ℃ (e.g., at least 20 ℃, 21 ℃,22 ℃, or 23 ℃), at least 4 ℃ (e.g., at least 5 ℃,10 ℃, or 15 ℃), at least-20 ℃ (e.g., at least-20 ℃, -15 ℃, -10 ℃, -5 ℃, or 0 ℃), or-80 ℃ (e.g., at least-80 ℃, -70 ℃, -60 ℃, -50 ℃, -40 ℃, or-30 ℃).

Or expressed refers to its activity that retains at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) relative to the initial activity of the CLP formulation or LNMP (e.g., at least 5%, 10%, or 15 ℃) over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90), relative to the CLP formulation or LNMP (e.g., at least 20 ℃, 21 ℃, 22 ℃, or 23 ℃), at least-20 ℃ (e.g., at least-20 ℃, -15 ℃, -10 ℃, -80 ℃), or at least 50 ℃, -80 ℃), optionally over a defined temperature range (e.g., at least 24 ℃, -25 ℃, 26 ℃, -27 ℃, -28 ℃, 29 ℃, or 30 ℃), or at least 50 ℃).

Or expression means that the CLP formulation or LNMP retains its particle size (i.e., does not increase in particle size) or has no more than 5% (e.g., no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 2-fold, 2.5-fold, or 3-fold increase relative to the initial size (e.g., at the time of manufacture or formulation) of the CLP or LNMP for a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days), optionally at a defined temperature range (e.g., a temperature of at least 24 ℃ (e.g., at least 24 ℃,25 ℃, 26 ℃, 27 ℃,28 ℃, 29 ℃, or 30 ℃), at least 20 ℃ (e.g., at least 20 ℃,21 ℃,22 ℃, or 23 ℃), at least 4 ℃ (e.g., at least 5 ℃,10 ℃, or 15 ℃), at least-20 ℃ (e.g., at least-20 ℃, -15 ℃, -10 ℃, -5 ℃, or 0 ℃), or-80 ℃ (e.g., at least-80 ℃, -70 ℃, -60 ℃, -50 ℃, -40 ℃, or-30 ℃).

In some embodiments, the stabilized CLP or LNMP formulation continues to encapsulate or remain associated with the exogenous peptide, polypeptide, or protein to which the CLP or LNMP formulation has been loaded, e.g., continues to encapsulate or remains associated with the exogenous peptide, polypeptide, or protein for at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, at least 90 days, or 90 days or more.

As used herein, the term "treatment" refers to administration of a pharmaceutical composition to an animal for prophylactic and/or therapeutic purposes. "preventing infection" refers to the prophylactic treatment of an animal that has not yet suffered a disease or disorder, but is susceptible to or otherwise at risk of suffering from a particular disease or disorder. "treating an infection" refers to administering a treatment to an animal that has already had a disease to improve or stabilize the animal's condition.

Drawings

FIG. 1 is a photograph showing the levels of nano-luciferases in mice treated with reconstituted LPMP (recPMP) oral (PO) derived from lemon, reconstituted LPMP formulated as C12-200 as ionizable lipids, and the photograph including nano-luciferases (nLuc) mRNA-nLuc-Flag and secreted nLuc.

Fig. 2 is a photograph showing the levels of nano-luciferases in mice treated with reconstituted LPMP (recPMP) oral (PO) derived from lemon, reconstituted LPMP formulated as C12-200 as ionizable lipids, and the photograph included nano-luciferases (nLuc) mRNA-nLuc-Flag and secreted nLuc in liver, stomach, colon, spleen, small intestine, mesenteric lymph node, pancreas and cecum, exposure time was 5 minutes.

FIG. 3 is a photograph showing the levels of nano-luciferases in mice treated with reconstituted LPMP (recPMP) oral (PO) derived from lemon, reconstituted LPMP formulated as ionizable lipids with C12-200, including liver, stomach, colon, spleen, small intestine, mesenteric lymph nodes, pancreas and cecum nano-luciferases (nLuc) mRNA-nLuc-Flag and secretory nLuc, exposure time 5 minutes.

FIG. 4 is an organ diagram showing nano-luciferase levels in mice treated with reconstituted LPMP (recPMP) oral (PO) derived from lemon, reconstituted LPMP formulated as ionizable lipids with C12-200, including nano-luciferase (nLuc) mRNA-nLuc-Flag and secreted nLuc in liver, stomach, colon, spleen, small intestine, mesenteric lymph nodes, pancreas and cecum.

Figures 5A-5F show the average irradiance (radance) in the liver (figure 5A), spleen (figure 5B), pancreas (figure 5C), MLN (figure 5D), gastrointestinal tract (GI) (figure 5E) and inguinal lymph node (figure 5F) of mice 24 hours after oral (PO) or jejunal (IJ) delivery of mRNA-LNP formulation using ionizable lipid 2243 (nLuc-Flag mRNA, IJ-10 μg, PO-200 μg). N=5/group compared to untreated mice (n=2).

Fig. 6A shows representative sections of Small Intestine (SI) of untreated tdmamato mice as negative control. FIG. 6B shows representative sections of SI 48 hours after jejunal administration of 2243 mRNA-LNP formulation (nLuc-Flag: CRE mRNA,15 μg; N=2) using ionizable lipid 2243, where light grey indicates transfected cells. FIG. 6C highlights the Peyer's Patch shown in FIG. 6B. Fig. 6D highlights the nap shown in fig. 6B.

Fig. 7 depicts anti-tnfα antibody level concentrations of 24h following administration in plasma of mice given 0% DSS and single intravenous administration of LNP/mRNA formulation using ionizable lipid 2243 (LNP 2243/anti-tnfα mRNA,0.6 mg/kg) and in plasma of mice given 2% DSS and single intravenous administration of LNP/mRNA formulation using ionizable lipid 2243 (LNP 2243/anti-tnfα mRNA,0.6 mg/kg). The control was plasma of untreated mice (0% DSS and no LNP 2243/anti-tnfα mRNA administered). N=5/group.

FIG. 8A shows the concentration of TNF alpha in feces three days after dosing in mice given 2% DSS and single intravenous administration of LNP/mRNA formulation using ionizable lipid 2243 (LNP 2243/anti-TNF alpha mRNA,0.6 mg/kg). The control was faeces of untreated mice (0% DSS and no LNP 2243/anti tnfα mRNA administered). Fig. 8B shows calprotectin (calprotectin) concentrations in feces 3 days post-dosing (day 3 post-DSS start), 24 h post-dosing (day 5 post-DSS start), and 2% DSS-dosing and 2% DSS-single intravenous administration of LNP/mRNA formulation using ionizable lipid 2243 (2243 LNP/anti-tnfa mRNA,0.6 mg/kg) in mice pre-dosing (day 3 post-DSS start), and mice administered 2% DSS and single intravenous administration of LNP/mRNA formulation using ionizable lipid 2243 (2243 LNP/anti-tnfa mRNA,0.6 mg/kg). The control was faeces of untreated mice (0% DSS and no LNP 2243/anti tnfa mRNA administered). N=5/group.

Fig. 9 shows the measured antibody concentrations (huIgG, ng/mL), n=5 in mouse plasma at days 0, 1,3, 7 and 14, respectively, after a single intravenous administration of an exemplary LPMP/mRNA formulation using ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg).

Fig. 10A shows the measured antibody concentration (huIgG, ng/mL) in plasma of 24h mice after a single intravenous administration using an exemplary LPMP/mRNA formulation of ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg), n=10. PBS was used as a control (n=1). Fig. 10B shows the measured antibody concentrations (huIgG, ng/mL), n=10 in 24h mice MLN and colon/cecum after a single intravenous administration using an exemplary LPMP/mRNA formulation of ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg). PBS was used as a control (n=1).

Figures 11A-11D show the antibody concentration (huIgG, ng/mL) in the colon/cecum content, mesenteric Lymph Nodes (MLN), small Intestine (SI) content and plasma of mice 24 hours after jejunal delivery with an exemplary LPMP/mRNA formulation of ionizable lipid 2272 (anti-PCSK 9 mRNA,65-75 μg). This experiment was an n=19/3 independent experiment compared to PBS (n=16/3 independent experiment). Statistically identified outliers are deleted.

Detailed Description

The inventors have surprisingly found that when the nano-luciferase (nluc-Flag) is administered intra-cellularly by oral gavage and the nano-luciferase is administered by secretion (secretory nLuc (nLuc-secreted)), extensive transfection in the digestive tract (extrahepatic tissue) and lymphoid organs results, and no activity is observed in the liver. nLuc-Flag and secretory nLuc were not detected in the liver, they were detected only in the gastrointestinal tract and associated lymphoid tissues. Furthermore, higher expression of secretory nLuc in the colon was observed compared to nluc-Flag.

A feature herein is that RNA therapeutics can be safely directed to the lymphatic transport system without delivery to the liver. This selectivity can be used to treat pancreatitis, IBD, ulcerative colitis, crohn's disease, colorectal cancer, and also for oral vaccines (norovirus, RSV, influenza, shingles (shingles), COVID, etc.).

In some embodiments, potentially unlocked RNA therapies include, but are not limited to, cancers of the gastrointestinal tract (i.e., gastric, pancreatic, colon), mRNA directed against cancer antigens, diphtheria toxin, anti-PD 1, chemokines, cytokines (IL 2, IL12, IL27, IFNg, IL 15), receptors, pancreatitis (mRNA for anti-inflammatory drugs), IBD (mRNA directed against IL10, anti-TNF, siRNA directed against TNFa, mRNA directed against inhibitory receptors), ulcerative colitis, crohn's disease, infectious diseases (viruses, bacteria & fungi), and microbiome modulation.

RNA compositions can be used to induce balanced immune responses against cancer, including cellular and humoral immunity, without the risk of, for example, the possibility of insertion mutation formation. These RNA compositions include one or more polynucleotides (e.g., RNA, such as mRNA or circRNA) encoding one or more tumor antigen polypeptides formulated in Complex Lipid Particles (CLPs). In some embodiments, the CLP is a lipid reconstituted natural messenger package (LNMP) comprising lipids extracted from one or more natural sources (i.e., natural lipids) and ionizable lipids. NMP is a lipid assembly produced in whole or in part from Extracellular Vesicles (EVs) of natural origin or segments, parts or extracts thereof. PMP is a lipid assembly produced in whole or in part by plant Extracellular Vesicles (EVs), or fragments, portions or extracts thereof. LNMP is NMP derived from lipid structures, wherein the lipid structures are destroyed and reassembled or reconstituted in the liquid phase. LPMP are PMPs derived from lipid structures, wherein the lipid structures are destroyed and reassembled or reconstituted in the liquid phase.

The present disclosure also includes a method for preparing an RNA composition comprising reconstructing a membrane comprising purified NMP lipids in the presence of ionizable lipids to produce LNMPs comprising ionizable lipids, and loading one or more polynucleotides encoding one or more polypeptides into the LNMPs.

Composite lipid particles and lipid reconstitution Natural messenger bag (LNMP)

Composite lipid particles

The Composite Lipid Particles (CLPs) described herein comprise a plurality of lipids, including structural lipids extracted from one or more natural sources (e.g., plants or bacteria). In some embodiments, the complex lipid particle is a Natural Messenger Package (NMP) incorporating a natural lipid extract. In some embodiments, the composite lipid particle is a lipid reconstituted natural messenger package (LNMP) incorporating a natural lipid extract and at least one exogenous ionizable lipid.

The complex lipid particle may further comprise at least exogenous ionizable lipids. The ionizable lipid has two or more of the following listed characteristics:

(i) At least 2 ionizable amines;

(ii) At least 3 lipid tails, wherein each lipid tail is at least 6 carbon atoms in length;

(iii) pKa from about 4.5 to about 7.5;

(V) The ratio N to P is at least 3.

The composite lipid particle may comprise from 10% w/w to 99% w/w of structural lipids derived from one or more lipid structures of natural origin, e.g. it may comprise at least 10% w/w, at least 20% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w or about 99% w/w of lipids derived from one or more lipid structures of natural origin.

In some embodiments, the composite lipid particle comprises about 10-95% w/w natural (e.g., plant, bacterial) lipids. For example, the complex lipid particles comprise about 25-95% w/w, about 30-95% w/w, about 35-95% w/w, about 40-95% w/w, about 45-95% w/w, about 50-95% w/w, about 55-95% w/w, about 60-95% w/w, about 65-95% w/w, about 70-95% w/w, about 75-95% w/w, about 80-95% w/w, or about 85-95% w/w of natural (e.g., plant, bacterial) lipids, based on the amount of total lipids in the complex lipid formulation.

The composite lipid particle may comprise 3-1000 lipids extracted from one or more natural (e.g., plant, bacterial) sources. In some embodiments, the natural source is a plant, plant extract, or a segment or portion of a plant. In some embodiments, the natural source is a bacterium, a fragment of a bacterium, or a portion of a bacterium. In some embodiments, the natural source is lemon. In some embodiments, the natural source is soybean. In other embodiments, the natural source is E.coli.

In some embodiments, the composite lipid particle comprises at least 10 natural lipids belonging to one or more classes selected from the group consisting of fatty acyl (FATTY ACYLS, FA), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterols. For example, the composite lipid particle comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids belonging to one or more classes selected from the group consisting of Fatty Acyl (FA), fatty acyl conjugates, phospholipids, glycerolipids, glycolipids, glycerophospholipids, sphingolipids, waxes, and sterols. In some embodiments, the composite lipid particle comprises lipids from at least two or at least three of these different classes.

In some embodiments, the complex lipid particle comprises at least 10 natural lipids belonging to one or more classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. For example, the composite lipid particle comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids belonging to one or more classes selected from the group consisting of glycerolipids, sphingolipids, and sterols. In some embodiments, the composite lipid particle comprises lipids from at least two or at least three of these different classes.

In some embodiments, the complex lipid particle may comprise one or more Glycerolipids (GL) or Glycerophospholipids (GP), which may further comprise a glycolipid.

In some embodiments, the complex lipid particle may comprise one or more glycerolipids selected from the group consisting of Phospholipids (PL), galactolipids, triacylglycerols (TG) and thiols (SL). In some embodiments, the CLP comprises one or more Glycerophospholipids (GP) selected from the group consisting of Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and Phosphatidylinositol (PI). In some embodiments, the composite lipid particle comprises one or more Sphingolipids (SPs) selected from the group consisting of a Sulfur Lipid (SL), a Glycosyl Inositol Phosphate Ceramide (GIPC), a glucosyl ceramide (GCer), a ceramide (Cer), and a free long-chain base (LCB). In some embodiments, the complex lipid particle comprises one or more phytosterols selected from the group consisting of campesterol, stigmasterol, β -sitosterol, Δ5-oat sterol, brassicasterol, oat sterol, 4-desmethylsterol, 4α -monomethyl sterol, Δ5-sterol, Δ7-sterol, α -spinasterol, Δ5, Δ7-sterol, phytosterol, and sitosterol.

CLP may comprise one or more natural lipids belonging to one or more classes or subclasses selected from the group consisting of fatty acids, fatty esters, fatty aldehydes, fatty amides, acyclic oxidized lipids, cyclic oxidized lipids, glycerolipids, monoacylglycerols, diacylglycerols, triacylglycerols, lactide (estolide), glycosylmonoacylglycerols, sulfoquinolone monoacylglycerols, monogalactomonoacylglycerols, digalactomonoacylglycerols, sulfoquinionodiacylglycerols, monogalactodiacylglycerols, digalactodiacylglycerols, glycosyldiacylglycerols, glycerophospholipids, phospholipids, lysophospholipids, phosphatidylinositol phosphates, n-modified phospholipids, oxy/oxidized phospholipids, sphingolipids, sphingosine bases, ceramides, phosphoceramides, glycosphingolipids, sterols, cholesterol esters, sterol esters, cholic acids, glycosides and acyl glycosides. The complex lipid particle may comprise one or more natural lipids belonging to one or more classes or subclasses selected from the group consisting of the classes or subclasses listed above. For example, the composite lipid particle comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids belonging to one or more classes or subclasses selected from the group consisting of the classes or subclasses listed above.

In some embodiments, the CLP comprises one or more natural lipids belonging to one or more subclasses selected from the group consisting of acyl diacylglycerol glucuronide, acyl hexose ceramide, acyl sterol glycoside, cholic acid, acyl carnitine, cholesterol ester, ceramide, cardiolipin, coenzyme Q, diacylglycerol, digalactodiglycol, diacylglycerol glucuronide, bislysocardiolipin, fatty acid ester of a hydroxy fatty acid, semi-bismonoacylglycerophosphate, hexosyl ceramide, lysophosphatidic acid, lysophosphatidylcholine, lysophosphatidylethanolamine, N-acyl-lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidylinositol, lysophosphatidylserine, monogalactodiglycol, lysocardiolipin, N-acyl ethanolamine, N-acyl glycine, N-acyl glycyl serine, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylethanol, phosphatidylglycerol, phosphatidylinositol, methanol phosphate, phosphatidylinositol, serine, phosphatidyl cholesterol, phosphatidyl glycerol, triglycidyl, phosphatidyl sulfato, glycerol, phosphatidyl sulfato-to-side, and triglycidyl sulfato-side. In some embodiments, the composite lipid particle comprises at least 10 natural (e.g., plant, bacterial) lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above. For example, the composite lipid particle comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

The complex lipid particle may comprise 10 or more natural lipids belonging to one or more subclasses selected from the group consisting of: acyl sterol glycosides, ceramides, digalactodiglycol diacylglycerol, diacylglycerol glucuronide, hemi-di-monoacylglycerol phosphate, hexosylceramide, lysophosphatidylcholine lysophosphatidylethanolamine, monogalactosyl diacylglycerol, phosphatidylcholine, phosphatidylethanolamine phosphatidyl ethanol, phosphatidyl glycerol, phosphatidyl inositol, sulfoquinitol diacylglycerol, and sterols. For example, the composite lipid particle comprises at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, at least 500, at least 600, at least 700, or at least 800 natural lipids belonging to one or more subclasses selected from the group consisting of the subclasses listed above.

The complex lipid particle may comprise natural lipids from at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 different classes or subclasses of lipids of natural origin. In some embodiments, the composite lipid particle comprises at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different classes or subclasses of natural lipids from a single natural source (e.g., from a plant source only or a bacterial source only). In some embodiments, CLP may comprise natural lipids from only one class or only one subclass of lipids of natural origin.

The identity (and class and subclass) and amount of lipids extracted from natural sources can be analyzed by lipidomic analysis by dissolving the lipid extract or complex lipid particles in a compatible solvent and analyzing by mass spectrometry (e.g., MS/MS). Other known methods, such as Charged Aerosol Detection (CAD) (e.g., HPLC-CAD, normal phase high performance liquid chromatography (NP-HPLC-CAD), or reversed phase high performance liquid chromatography (RP-HPLC-CAD)), may also be used.

The composite lipid particle may comprise all or part of the lipid material(s) present in the lipid structure from a particular natural source, e.g. it may comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or almost 100% of the lipid material(s) present in the lipid structure from a particular natural source.

The complex lipid particles may comprise reduced or minimized proteinaceous material that is endogenous to the one or more natural sources. For example, the composite lipid particle may comprise less than 50% w/w, less than 45% w/w, less than 40% w/w, less than 35% w/w, less than 30% w/w, less than 25% w/w, less than 20% w/w, less than 15% w/w, less than 10% w/w, less than 9% w/w, less than 8% w/w, less than 7% w/w, less than 6% w/w, less than 5% w/w, less than 4% w/w, less than 3% w/w, less than 2% w/w, less than 1% w/w, less than 0.5% w/w, less than 0.1% w/w, or be substantially free of proteinaceous material that is endogenous to the one or more natural sources. In some cases, the lipid bilayer of the composite lipid particle does not contain a protein. To calculate the% w/w of residual proteinaceous matter endogenous to the one or more natural sources, the protein concentration is divided by the concentration of the natural lipid extract and then multiplied by 100. Or% w/w is calculated as the percentage of total protein mass endogenous to the one or more natural sources based on total lipid extract mass.

The complex lipid particles may comprise reduced or minimized residual dsDNA material that is endogenous to the one or more natural sources. For example, the composite lipid particle may comprise less than 15% w/w, less than 10% w/w, less than 5% w/w, less than 1% w/w, less than 0.5% w/w, less than 0.1% w/w, less than 0.05% w/w, less than 0.01% w/w, less than 0.005% w/w, less than 0.001% w/w, or be substantially free of residual dsDNA material that is endogenous to the one or more natural sources. In some cases, the lipid bilayer of the composite lipid particle does not contain residual dsDNA. To calculate the% w/w of residual dsDNA material endogenous to the one or more natural sources, the total adjusted dsDNA concentration is divided by the concentration of the natural lipid extract and then multiplied by 100. Or% w/w is calculated as the percentage of total residual dsDNA mass endogenous to the one or more natural sources based on the mass of the total lipid extract.

In some embodiments, the composite lipid particle further comprises a synthetic structural lipid, such as a neutral lipid. In some embodiments, the structural lipid component of the composite lipid particle may comprise 10% w/w to 99% w/w of structural lipids derived from synthetic lipid structures, e.g., it may comprise at least 10% w/w, at least 20% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, at least 95% w/w, or about 99% w/w of lipids derived from synthetic lipid structures.

In addition to the exogenous ionizable lipid, the composite lipid particle may further comprise at least two other exogenous lipids. The composite lipid particle may comprise at least 1% w/w, at least 2% w/w, at least 5% w/w, at least 10% w/w, at least 15% w/w, at least 20% w/w, at least 25% w/w, at least 30% w/w, at least 40% w/w, at least 50% w/w, at least 60% w/w, at least 70% w/w, at least 80% w/w, at least 90% w/w, or about 95% w/w of the exogenous lipid. Exemplary exogenous lipids include ionizable lipids, synthetic structural lipids, sterols, and PEG-lipid conjugates. The complex lipid particle may also comprise at least two exogenous lipids. In some embodiments, the composite lipid particle comprises an ionizable lipid, a sterol, and a PEG-lipid conjugate. Additional exogenous lipids suitable for inclusion in the composite lipid particle are described herein below.

In some embodiments, the CLP contains a natural lipid comprising a fatty acid-derived tail that is:

About 5% to 20% fatty acid 16:0

About 0% to 10% fatty acids 18:1 (C9)

About 0% to 10% fatty acids 18:1 (C7)

About 5% to 30% fatty acids 18:2

About 2% to 20% fatty acids 18:3.

In some embodiments, the CLP contains a Phosphatidylcholine (PC) lipid comprising a fatty acid-derived tail that is:

about 10% to 20% fatty acid 16:0

About 2% to 5% fatty acid 18:0

About 7% to 15% fatty acids 18:1

About 50% to 75% fatty acids 18:2

About 2% to 10% fatty acids 18:3.

In some embodiments, the CLP contains a Phosphatidylethanolamine (PE) lipid comprising a fatty acid-derived tail that is:

about 0.25% to 5% fatty acid 14:0

About 25% to 45% fatty acid 16:0

About 5% to 15% fatty acid 16:1

About 10% to 25% fatty acid 17:0

About 25% to 45% fatty acid 18:1

About 2% to 7% fatty acids 19:0.

In some embodiments, CLP contains natural lipids belonging to the subclasses phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin, comprising:

About 50 to 75 wt/wt% Phosphatidylethanolamine (PE)

About 15 to 30 wt/wt% Phosphatidylglycerol (PG)

About 5 to 15 weight/weight% Cardiolipin (CL).

In some embodiments, CLP contains a natural lipid comprising:

about 10 to 50 weight/weight% Phosphatidylcholine (PC)

About 5 to 50 wt/wt% Phosphatidylethanolamine (PE)

About 0 to 15 wt/wt% Triacylglycerols (TG)

About 5 to 35 weight/weight% hexosylceramide (HexCer)

About 0 to 5 wt/wt% Phosphatidylglycerol (PG)

About 0 to 7 wt/wt% Phosphatidylserine (PS)

About 0 to 10 wt/wt% Phosphatidylinositol (PI)

About 0 to 5 weight/weight% Cardiolipin (CL).

In some embodiments, the composite lipid particle comprises less than 12% w/w chloroplasts endogenous to the one or more natural sources. In some embodiments, the composite lipid particle comprises less than 20% w/w, less than 15% w/w, less than 10% w/w, less than 5% w/w, less than 1% w/w, less than 0.5% w/w, or less than 0.1% w/w chloroplasts endogenous to the one or more natural sources.

In some embodiments, the composite lipid particle comprises less than 5% w/w exogenous antioxidant.

In some embodiments, CLP contains natural lipids comprising about 0 to 20 wt/wt% Cardiolipin (CL).

Natural messenger bag (NMP)

The plurality of NMPs in the modified NMP formulation may be loaded with the exogenous peptide, polypeptide, or protein such that at least 5%, at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 95% of the plurality of NMPs encapsulate the exogenous peptide, polypeptide, or protein. In some embodiments, the NMP is derived from an arthropod, fungus, archaea, plant, or bacterium. For example, one example of NMP derived from a plant source is plant NMP, which may be referred to as PMP, which is a lipid (e.g., lipid bilayer, monolayer, or multilayer structure) structure comprising a plant EV, or a segment, portion, or extract thereof (e.g., lipid extract). Additional description of PMP can be found in PCT application No. PCT/US22/47107 filed on 10 month 19 2022, the entire contents of which are incorporated herein by reference, in the section "Plant Messenger Package (PMP)".

NMP may comprise an arthropod, plant, fungus, archaea or bacterial EV, or a segment, fraction or extract thereof, wherein EV has a diameter of about 5-2000 nm. For example, NMP may comprise EV or a segment, fraction or extract thereof having an average diameter of about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1250 nm, about 1250-1500 nm, about 1500-1750 nm, or about 1750-2000 nm. In some cases, NMP comprises an arthropod, plant, fungus, archaea, or bacterium EV, or a segment, fraction, or extract thereof, having an average diameter of about 5-1400 nm, 5-950 nm, about 5-900 nm, about 5-850 nm, about 5-800 nm, about 5-750 nm, about 5-700 nm, about 5-650 nm, about 5-600 nm, about 5-550 nm, about 5-500 nm, about 5-450 nm, about 5-400 nm, about 5-350 nm, about 5-300 nm, about 5-250 nm, about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm, or about 5-nm. In certain instances, the arthropod, plant, fungus, archaea, or bacterial EV, or a segment, portion, or extract thereof, has an average diameter of about 50-200 nm. In some cases, the EV or a segment, fraction or extract thereof has an average diameter of about 50-300 nm. In some cases, the EV or a segment, fraction or extract thereof has an average diameter of about 200-500 nm. In some cases, the EV or a segment, fraction or extract thereof has an average diameter of about 30-150 nm.

In some cases, NMP may comprise an arthropod, plant, fungus, archaea, or bacterium EV, or a segment, portion, or extract thereof, having an average diameter of at least 5nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, or at least 1300. In some cases, NMP comprises arthropod, fungus, archaea, or bacteria EV, or a segment, fraction, or extract thereof, having an average diameter of less than 1400 nm, less than 1000 nm, less than 950 nm, less than 900 nm, less than 850 nm, less than 800 nm, less than 750 nm, less than 700 nm, less than 650 nm, less than 600 nm, less than 550 nm, less than 500 nm, less than 450 nm, less than 400 nm, less than 350 nm, less than 300 nm, less than 250 nm, less than 200 nm, less than 150 nm, less than 100 nm, or less than 50 nm. The particle size of the EV, or a segment, fraction or extract thereof, may be measured using a variety of standard methods in the art (e.g., dynamic light scattering methods).

In some cases, NMP may comprise arthropods, plants, fungi, archaea, or bacterial EV, or segments thereof, a fraction or extract having an average surface area of 77 nm ² to 3.2 x10 ⁶nm² (e.g., ,77-100 nm²、100-1000 nm²、1000-1x10⁴nm²、1x10⁴-1x10⁵nm²、1x10⁵-1x10⁶nm²、 or 1x10 ⁶-3.2x10⁶nm²). In some cases, the NMP may comprise an arthropod, fungus, archaea, or bacteria EV, or a segment, fraction, or extract thereof, having an average volume of 65 nm ³ to 5.3x10 ⁸nm³ (e.g., ,65-100 nm³、100-1000 nm³、1000-1x10⁴nm³、1x10⁴-1x10⁵nm³、1x10⁵-1x10⁶nm³、1x10⁶-1x10⁷nm³、1x10⁷-1x10⁸nm³、1x10⁸-5.3x10⁸nm³）. in some cases, NMP may comprise an arthropod, A plant, fungus, archaea or bacterium EV, or a segment, part or extract thereof, having an average surface area of at least 77 nm ² (e.g., at least 77 nm ², at least 100 nm ², at least 1000 nm ², At least 1x10 ⁴nm², at least 1x10 ⁵nm², at least 1x10 ⁶nm², or at least 2x10 ⁶nm²). In some cases, the NMP may comprise an arthropod, fungus, archaea, or bacteria EV, or a segment, fraction, or extract thereof, having an average volume of at least 65 nm ³ (e.g., at least 65 nm ³, at least 100 nm ³, At least 1000 nm ³, at least 1x10 ⁴nm³, at least 1x10 ⁵nm³, at least 1x10 ⁶nm³, At least 1x10 ⁷nm³, at least 1x10 ⁸nm³, at least 2x10 ⁸nm³, at least 3x10 ⁸nm³, At least 4x10 ⁸nm³, or at least 5x10 ⁸nm³.

In some cases, NMP may have the same size as arthropods, plants, fungi, archaea, or bacteria EV, or segments, extracts, or portions thereof. Or NMP may have a different size than the original EV that produced NMP. For example, NMP may have a diameter of about 5 to 2000 nm a. For example, the average diameter of NMP may be about 5-50 nm, about 50-100 nm, about 100-150 nm, about 150-200 nm, about 200-250 nm, about 250-300 nm, about 300-350 nm, about 350-400 nm, about 400-450 nm, about 450-500 nm, about 500-550 nm, about 550-600 nm, about 600-650 nm, about 650-700 nm, about 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1200 nm, about 1200-1400 nm, about 1400-1600 nm, about 1600-1800 nm, or about 1800-2000 nm. In some cases, the average diameter of NMP may be at least 5 nm, at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm, at least 250 nm, at least 300 nm, at least 350 nm, at least 400 nm, at least 450 nm, at least 500 nm, at least 550 nm, at least 600 nm, at least 650 nm, at least 700 nm, at least 750 nm, at least 800 nm, at least 850 nm, at least 900 nm, at least 950 nm, at least 1000 nm, at least 1200 nm, at least 1400 nm, at least 1600 nm, at least 1800 nm, or about 2000 nm. The particle size of NMP can be measured using a variety of standard methods in the art (e.g., dynamic light scattering methods). In some cases, the size of NMP is determined after loading with heterologous functional agents or after other modifications to NMP.

In some cases, the average surface area of NMP may be 77 nm ² to 1.3x10 ⁷nm² (e.g., ,77-100 nm²、100-1000 nm²、1000-1x10⁴nm²、1x10⁴-1x10⁵nm²、1x10⁵-1x10⁶nm²、 or 1x10 ⁶-1.3x10⁷nm²). In some cases, the average volume of NMP may be 65 nm ³ to 4.2x10 ⁹nm³ (e.g., ,65-100 nm³、100-1000 nm³、1000-1x10⁴nm³、1x10⁴-1x10⁵nm³、1x10⁵-1x10⁶nm³、1x10⁶-1x10⁷nm³、1x10⁷-1x10⁸nm³、1x10⁸-1x10⁹nm³、 or 1x10 ⁹-4.2x10⁹nm³). In some cases, the NMP has an average surface area of at least 77 nm ² (e.g., at least 77 nm ², at least 100 nm ², at least 1000 nm ², At least 1x10 ⁴nm², at least 1x10 ⁵nm², at least 1x10 ⁶nm², or at least 1x10 ⁷nm²). In some cases, the average volume of NMP is at least 65 nm ³ (e.g., at least 65 nm ³, at least 100nm ³, at least 1000 nm ³, At least 1x10 ⁴nm³, at least 1x10 ⁵nm³, at least 1x10 ⁶nm³, at least 1x10 ⁷nm³, At least 1x10 ⁸nm³, at least 1x10 ⁹nm³, at least 2x10 ⁹nm³, at least 3x10 ⁹nm³, Or at least 4x10 ⁹nm³,).

In some cases, NMP may include whole arthropods, plants, fungi, archaea, or bacterial EVs. In some embodiments, NMP may comprise a non-plant natural source, such as algae or animal derived organ EV, or a segment, portion or extract thereof. Or NMP may comprise a full surface area segment, portion, or extract of a vesicle of an EV (e.g., comprising less than 100% (e.g., less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%) of a full surface area segment, portion, or extract of a vesicle). The segments, portions or extracts may be of any shape, such as circumferential segments, spherical segments (e.g. hemispheres), curvilinear segments, linear segments or flattened segments. In the case where the segment is a spherical segment of a vesicle, the spherical segment may represent a spherical segment resulting from splitting a spherical vesicle along a pair of parallel lines, or a spherical segment resulting from splitting a spherical vesicle along a pair of non-parallel lines. Thus, the plurality of NMPs may comprise a plurality of complete EVs, a plurality of EV segments, portions or extracts, or a mixture of complete EVs and EV segments. Those skilled in the art will appreciate that the ratio of complete EVs to segment EVs will depend on the particular separation method used. For example, grinding or blending an arthropod, fungus, plant, archaea, or bacterium, or portion thereof, can produce NMP that contains a higher percentage of EV segments, portions, or extracts than a non-destructive extraction method (e.g., vacuum infiltration).

In the case where NMP comprises segments, portions or extracts of arthropods, fungi, archaea or bacteria EV, the average surface area of the EV segments, portions or extracts may be less than the average surface area of the intact vesicles, e.g., the average surface area is less than 77 nm²、100 nm²、1000 nm²、1x10⁴nm²、1x10⁵nm²、1x10⁶nm²、 or 3.2x10 ⁶nm². In some cases, the EV segment, fraction, or extract has a surface area of less than 70 nm ²、60 nm²、50 nm²、40 nm²、30 nm²、20 nm², or 10 nm ²). In some cases, NMP may comprise arthropods, fungi, archaea or bacteria EV, or segments, fractions or extracts thereof, having an average volume that is less than the average volume of intact vesicles, e.g., an average volume that is less than 65 nm³、100 nm³、1000 nm³、1x10⁴nm³、1x10⁵nm³、1x10⁶nm³、1x10⁷nm³、1x10⁸nm³、 or 5.3x10 ⁸nm³.

In the case where the NMP comprises an extract of an arthropod, plant, fungus, archaea or bacteria EV, for example, where the NMP comprises lipids extracted from the EV (e.g., with chloroform or ethanol), the NMP may comprise at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more than 99% of the lipids extracted from the arthropod, fungus, archaea or bacteria EV (e.g., with chloroform or ethanol). The NMP in the plurality may include arthropod, plant, fungal, archaea or bacterial EV segments and/or EV extracted lipids or mixtures thereof.

NMP production

NMP may be produced by an arthropod, fungus, plant, archaea or bacterium EV, or a segment, part or extract thereof (e.g., a lipid extract), which naturally occurs in an arthropod, fungus, plant, archaea or bacterium, or a part thereof (including tissues or cells). In some embodiments, NMP may comprise a non-plant natural source, such as algae or animal derived organ EV, or a segment, portion or extract thereof. An exemplary method for producing NMP includes (a) providing an initial sample from a source or portion thereof, wherein the source or portion thereof comprises EV, and (b) separating a crude NMP fraction from the initial sample, wherein the level of at least one contaminant or undesirable component of the crude NMP fraction from the source or portion thereof is reduced relative to the level in the initial sample. The method may further comprise a further step (c) comprising purifying the crude NMP fraction, thereby producing a plurality of pure NMPs, wherein the level of at least one contaminant or undesirable component from an arthropod, fungus, archaea, or bacterium, or portion thereof, of the plurality of pure NMPs is reduced relative to the level in the crude EV fraction. Each generation step is discussed in further detail below.

For example, PMP may be produced from a plant EV, or a segment, part or extract thereof (e.g., a lipid extract), which naturally occurs in a plant or a part thereof (including plant tissue or plant cells). An exemplary method for producing PMP includes (a) providing an initial sample from a plant or portion thereof, wherein the plant or portion thereof comprises an EV, and (b) separating a crude PMP fraction from the initial sample, wherein a level of at least one contaminant or undesirable component of the crude PMP fraction from the plant or portion thereof is reduced relative to a level in the initial sample. The method may further comprise a further step (c) comprising purifying the crude PMP fraction, thereby producing a plurality of pure PMPs, wherein the level of at least one contaminant or undesired component from the plant or portion thereof of the plurality of pure PMPs is reduced relative to the level in the crude EV fraction. Each of the generating steps is discussed in further detail below. Exemplary methods for separating and purifying NMP (e.g., plant NMP, PMP) are found, for example, in Rutter and Innes,Plant Physiol. 173(1): 728-741, 2017;Rutter et al,Bio. Protoc. 7(17): e2533, 2017;Regente et al,J of Exp. Biol. 68(20): 5485-5496, 2017;Mu et al, Mol. Nutr. Food Res., 58, 1561–1573, 2014 and REGENTE ET AL, FEBS letters, 583:3363-3366, 2009, each of which is incorporated herein by reference. Additional description of the generation of PMPs can be found in PCT application No. PCT/US22/47107 filed on 10/19 2022, the entire contents of which are incorporated herein by reference.

For example, a plurality of NMPs may be isolated from an arthropod, fungus, plant, archaea, or bacterium by a method comprising (a) providing an initial sample from a source or portion thereof, wherein the source or portion thereof comprises an EV, (b) separating a crude NMP fraction from the initial sample, wherein the level of at least one contaminant or non-desired component of the crude NMP fraction from the arthropod, fungus, plant, archaea, or bacterium, or portion thereof, relative to the level in the initial sample, is reduced (e.g., reduced by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100%), and (c) purifying the crude NMP fraction, thereby producing a plurality of pure NMPs, wherein the level of at least one contaminant or non-desired component of the plurality of pure NMPs from the source or portion thereof is reduced (e.g., reduced by at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 99%, 80%, 98%, 90%, 100%) relative to the level in the crude fraction.

NMP provided herein may include arthropods, fungi, archaea, or bacterial EVs isolated from a variety of sources, or segments, portions, or extracts thereof.

For example, the plant NMP, PMP may comprise plant EV produced by a variety of plants or a segment, part or extract thereof. PMP can be produced from plants of any genus (vascular or vascular-free), including but not limited to angiosperms (monocots and dicots), gymnosperms, ferns, selaginella, equisetum, gymnosperms, pinus, algae (e.g., unicellular or multicellular algae such as protopigment organisms (ARCHAEPLASTIDA)) or bryophytes. In some cases, PMP may be produced using vascular plants such as monocots or dicots or gymnosperms. For example, PMPs may be produced using alfalfa, apple, arabidopsis (Arabidopsis), banana, barley, brassica species (e.g., arabidopsis (Arabidopsisthaliana) or canola (Brassica napus)), canola, castor bean, chicory, chrysanthemum, clover, cocoa, coffee, cotton, cottonseed, corn, broccoli, cranberry, cucumber, dendrobium, yam, eucalyptus, fescue, flax, gladiolus, liliaceae, linseed, millet, melon, mustard, oat, oil palm, canola, papaya, peanut, pineapple, ornamental, bean, potato, rapeseed, rice, rye, ryegrass, safflower, sesame, sorghum, soybean, beet, sugarcane, sunflower, strawberry, tobacco, tomato, turf grass, wheat, or vegetable crops such as lettuce, celery, broccoli, cauliflower, cucurbits; fruit trees and nut trees such as apples, pears, peaches, oranges, grapefruits, lemons, limes, almonds, pecans, walnuts, hazelnuts, vines such as grapes, kiwi fruits, hops, fruit shrubs and thorns such as raspberries, blackberries, gooseberries, currants, forest trees such as white wax trees, pine trees, fir, maple, oaks, chestnut trees, poplars, alfalfa, canola, castor bean, corn, cotton, broccoli, flax, linseed, mustard, oil palm, rape, peanuts, potatoes, rice, safflower, sesame, soybeans, beets, sunflowers, tobacco, tomatoes, or wheat.

PMP (i.e., plant NMP) may be produced using whole plants (e.g., whole flowers or seedlings) or alternatively from one or more plant parts (e.g., leaves, seeds, roots, fruits, vegetables, pollen, phloem juice, or xylem juice). For example, PMPs can be produced using bud vegetative organs/structures (e.g., leaves, stems, or tubers), roots, flowers, and floral organs/structures (e.g., pollen, bracts, sepals, petals, stamens, carpels, anthers, or ovules), seeds (including embryos, endosperm, or seed coats), fruits (mature ovaries), juices (e.g., phloem or xylem juice), plant tissue (e.g., vascular tissue, root tissue, tumor tissue, etc.), and cells (e.g., single cells, protoplasts, embryos, callus, guard cells, egg cells, etc.), or progeny thereof. For example, the isolating step may involve (a) providing a plant or part thereof. In some examples, the plant part is an arabidopsis leaf. The plant may be at any stage of development. For example, PMP may be produced using seedlings, for example, seedlings of 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks old (e.g., arabidopsis seedlings). Other exemplary PMPs may include PMPs produced using roots (e.g., ginger root), fruit juices (e.g., grapefruit juice), vegetables (e.g., broccoli), pollen (e.g., olive pollen), phloem juice (e.g., arabidopsis phloem juice), or xylem juice (e.g., tomato plant xylem juice). In some embodiments, PMP is produced from algae or lemon.

NMP may be isolated from any genus of arthropod, fungus, archaea or bacteria, including but not limited to crab, crayfish, shrimp, spider, scorpion, cricket, grasshopper, beetle, horse land, ticks, mites, centipede, ant, wasp, dragonfly, fly, mosquito, other insects and crustaceans, yeast, mushroom, puffball, phallus, boletus, soot (smut), fishy soot (bunt), eave (bracket fungi), glial (jelly fungi), toxic fungus (toadstools), mold (molds), rust (rusts), Arisaema (EARTH STARS), oletum Trogopterori (CHANTERELLES), clavipita (ergot), pyricularia (pyrolobus), klebsiella philippica (picrophilus), methanogen (methanogens), rhizoctonia (crenarchaeota) the plant extracts comprise a nanometer archaea (nanoarchaeota), a fuel coccus (ignicoccus), an archaea (cenarchaeum), a halophil (halophiles), an Escherichia, Acinetobacter (Acinetobacter), agrobacterium (Agrobacterium), anabaena (Anabaena), anaplasma (ANAPLASMA), aquifex, azoarcus (Azoarcus), azospirillum (Azospiralum), azotobacter (Azotobacter), ballpass (Bartonella), barthogon (Bordetella), rhizobium chroophoroides (Bradyrhizobium), brucella (Brucella), Brucella (Buchnera), burkholderia (Burkholderia), bacillus phloem (Candidatus), chromobacterium (Chromobacterium), coxiella (Coxiella), chlorella (Crocosphaera), dechloromonas (Dechloromonas), dehalobacter (Desulfitobacterium), desulfobacillus (Desulfotalea), erwinia (Erwinia), francisella (FRANCISELLA), Fusobacterium (Fusobacterium), myxobacterium (Gloeobacter), gluconobacter (Gluconobacter), helicobacter (Helicobacter), legionella (Legionella), massa Medicata Fermentata (Magnetospirillum), megazobium mesorhizogenes (Mesorhizobium), methylobacillus (Methylobacterium), methylococcus (Methylococcus), neisseria (Neisseria), nitrosomonas (Nitrosomonas), Nostoc (Nostoc), luminous bacillus (Photobacterium), polish rod bacteria (Photohabdus), phyllobacterium (Phyllobacterium), polar unit cell (Polaromonas), protogreen chlorella (Prochlorococcus), pseudomonas (Pseudomonas), leng Ganjun (Psychrobacter), ralstonia (Ralstonia), red living bacteria (Rubrivivax), salmonella (Salmonella), and their use, Shewanella (Shewanella), shigella (Shigella), sinorhizobium (Sinorhizobium), synechococcus (Synechococcus), synechocystis (Synechocystis), thermococcus (Thermotoga), thermus (Thermus), thiobacillus (Thermobacillus), shu Maozao (Trichodesmium), vibrio (Vibrio), wiguelover (Wigglesworthia), Wo Linshi (Wolinella), xanthomonas (Xanthomonas), wood-forming bacteria (Xylella), yersinia (Yersinia), bacillus (Bacillus), bifidobacterium (Bifidobacterium), clostridium (Clostridium), corynebacterium (Corynebacterium), deinococcus (Deinococcus), enterococcus (Enterococcus), microbacterium (Exiguobacterium), geobacillus (Geobacillus), Lactobacillus, listeria Leuconostoc (Leuconostoc) Mushroom (Moorella), bacillus megaterium (Oceanobacillus), rhizobium (Rhizobium) Rickettsia (Rickettsia), staphylococci (Staphylococcus), streptococci (Streptococcus), symbiotic bacilli (Symbiobacterium) or thermo anaerobic bacilli (thermo anaerobacter).

NMP may be produced by whole arthropods, fungi, archaea or bacteria (e.g., whole insects, arachnids, crustaceans, single cells of fungi or archaea or bacteria), or alternatively by one or more source fractions (e.g., segments, organs, eggs, spores, mycelia, tissues, membranes or cell walls). For example, NMP may be produced from organ/structure/tissue/cell cultures (e.g., body segments, appendages, organs, eggs, exoskeletons, embryos, spores, mycelium, hyphae, thalli, suspension cultures, cell walls, inner or outer membranes, gametophytes, sporophytes, polymerases, glycerol-ether lipids, metabolites, flagella, pili, ribosomes, or organelles) or their offspring. The source may be at any stage of development. In some embodiments, NMP is produced by insects or fungi (e.g., cricket, yeast, or mushrooms). In some embodiments, NMP is produced by bacteria or archaea (e.g., e.coli). In some embodiments, NMP is produced from algae (e.g., kelp or chlorella). In some embodiments, NMP is produced by an animal organ (e.g., brain or blood).

NMP can be produced by a variety of methods from plants, arthropods, fungi, archaea or bacteria, or parts thereof. Any method that allows for the release of the EV-containing fraction of the source or other extracellular fraction (e.g., cell culture medium) containing NMP that contains secreted EVs is suitable for use in the present method. EV may be isolated from the source or source portion by destructive (e.g., milling or blending) or non-destructive (washing or vacuum infiltration) methods. For example, plants, arthropods, fungi, archaea or bacteria, or portions thereof, may be vacuum infiltrated ground, milled, blended, or a combination thereof to separate the EV from the source or source portion, thereby producing NMP. For example, the separation step may comprise (b) separating a crude NMP fraction from an initial sample (e.g., a plant, arthropod, fungus, archaea, or bacterium, or portion, or a sample derived from a plant, arthropod, fungus, archaea, or bacterium, or portion), wherein the level of at least one contaminant or undesired component of the crude NMP fraction from the source, or portion thereof, is reduced relative to the level in the initial sample, wherein the separation step comprises vacuum infiltration of the plant, arthropod, fungus, archaea, or bacterium (e.g., separation of buffer with vesicles) to release and collect the desired fraction. Or the separation step may comprise (b) milling or blending the source to release EV, thereby producing NMP.

Upon isolation of plants, arthropods, fungi, archaea or bacteria EV, thereby producing NMP, NMP may be separated or collected into a crude NMP fraction. For example, the separation step may involve separating the plurality of NMP into a crude NMP fraction using centrifugation (e.g., differential centrifugation or ultracentrifugation) and/or filtration to separate the NMP-containing fraction from large contaminants (including tissue fragments, cells, or organelles). Thus, the crude NPM fraction will have a reduced amount of large contaminants, including, for example, tissue fragments, cells, or organelles (e.g., nuclei, mitochondria, etc.), as compared to the initial sample from the source or source fraction.

The crude NMP fraction may be further purified by additional purification methods to yield a variety of pure NMPs. For example, the crude NMP fraction may be separated from other source components by ultracentrifugation, such as using a density gradient (iodixanol or sucrose), size exclusion, and/or other methods of removing aggregated components (e.g., precipitation or size exclusion chromatography). The level of contaminants or undesirable components (e.g., one or more non-NMP components, e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures), nuclei, cell wall components, organelles, or combinations thereof) from the source of the resulting pure NMP may be reduced relative to one or more fractions produced in the early separation step, or relative to a pre-set threshold level, e.g., commercial release profile. For example, the level of the source organelle or cell wall component of pure NMP may be reduced (e.g., reduced by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%; or reduced by about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) relative to the level in the initial sample. In some cases, the pure NMP is substantially free (e.g., has undetectable levels) of one or more non-NMP components, such as protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures), nuclei, cell wall components, organelles, or combinations thereof. Further examples of release and separation steps can be found in WO 2021/04301. The concentration of NMP may be, for example, 1x10⁹、5x10⁹、1x10¹⁰、5x10¹⁰、5x10¹⁰、1x10¹¹、2x10¹¹、3x10¹¹、4x10¹¹、5x10¹¹、6x10¹¹、7x10¹¹、8x10¹¹、9x10¹¹、1x10¹²、2x10¹²、3x10¹²、4x10¹²、5x10¹²、6x10¹²、7x10¹²、8x10¹²、9x10¹²、1x10¹³、 or more than 1x10 ¹³ NMP/mL.

For example, protein aggregates can be removed from the isolated PMP. For example, the separated NMP solution may be removed through a range of pH (e.g., as measured using a pH probe) to precipitate protein aggregates in the solution. The pH may be adjusted to, for example, pH 3, pH 5, pH 7, pH 9 or pH 11 with the addition of, for example, sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it may be filtered to remove particulates. Alternatively, the separated NMP solution may be flocculated using the addition of a charged polymer such as Polymin-P or Praestol 2640. Briefly, polymin-P or Praestol 2640,2640 was added to the solution and mixed with an impeller. The solution may then be filtered to remove particulates. Or the aggregate may be dissolved by increasing the salt concentration. For example, naCl may be added to the separated NMP solution until it reaches, for example, 1 mol/L. The solution may then be filtered to isolate NMP. Or by increasing the temperature to dissolve the aggregates. For example, the separated NMP may be heated with mixing until the solution reaches a uniform temperature of, for example, 50 ℃ for 5 minutes. The NMP mixture may then be filtered to isolate NMP. Alternatively, soluble contaminants may be separated from the NMP solution by size exclusion chromatography according to standard procedures, wherein NMP is eluted in a first fraction and proteins and ribonucleoproteins and some lipoproteins are subsequently eluted. The efficiency of protein aggregate removal can be determined by BCA/Bradford protein quantification to measure and compare protein concentration before and after protein aggregate removal. In some embodiments, protein aggregates are removed before the exogenous peptide, polypeptide, or protein is encapsulated by NMP. In other embodiments, protein aggregates are removed after the exogenous peptide, polypeptide, or protein is encapsulated by NMP.

In some embodiments, the preparation of NMP from a natural source is performed by an ethanol extraction process. In some aspects, a 3:2 ethyl acetate to ethanol solvent aids in extraction. In some embodiments, the preparation of NMP from a natural source is performed by a modified Matyash extraction process. In some aspects, a 1:2 MeOH in MTBE solvent aids in extraction.

Any of the production methods described herein can be supplemented with any quantitative or qualitative methods known in the art to characterize or identify NMP at any step of the production process. NMP can be characterized by a variety of analytical methods to estimate NMP yield, NMP concentration, NMP purity, NMP composition, or NMP size. In some cases, methods (e.g., mass spectrometry) may be used to identify EV markers present on NMP, to facilitate analysis and characterization of NMP fractions, NMP may additionally be labeled or stained, for example, with 3,3' -dihexyloxycarbocyanine (DIOC ₆) (fluorescent lipophilic dye), PKH67 (SIGMA ALDRICH), alexa Fluor 488 (Thermo FISHER SCIENTIFIC), or DyLight ^TM (Thermo Fisher) in the absence of complex forms of nanoparticle tracking, such relatively simple methods quantify total membrane content, and may be used to indirectly measure NMP concentration （Rutter and Innes,Plant Physiol. 173(1): 728-741, 2017;Rutter et al,Bio. Protoc. 7(17): e2533, 2017）. and to accurately measure the size distribution of NMP or to accurately assess the flow resistance of the nanoparticles.

During production, NMP may optionally be prepared such that the concentration of NMP is increased (e.g., by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more than 100%, or by about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold or more than 100-fold) relative to the EV level in the control or initial sample. The isolated NMP may comprise any of about 0.1% to about 100%, for example about 0.01% to about 100%, about 1% to about 99.9%, about 0.1% to about 10%, about 1% to about 25%, about 10% to about 50%, about 50% to about 99% of the NMP composition. In some cases, the compositions described herein include at least any of 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or higher NMP, e.g., as measured by weight/volume, NMP protein composition percentage and/or lipid composition percentage (e.g., by measuring fluorescently labeled lipids). In some cases, the concentrate is used as a commercial product, for example, the end user may use a significantly lower concentration of the active ingredient as a diluent. In some embodiments, the compositions described herein are formulated as NMP concentrate formulations, e.g., ultra low volume concentrate formulations. In some embodiments, the concentration of NMP in the composition is effective to increase the adaptation of an organism, such as a plant, animal, insect, bacteria, or fungus. In other aspects, the concentration of NMP in the composition is effective to reduce the adaptation of an organism, such as a plant, animal, insect, bacteria, or fungus.

NMP may be produced by a variety of arthropods, fungi, plants, archaea or bacteria, or one or more parts thereof (e.g., fragments, organs, eggs, spores, mycelia, tissues, membranes, or cell walls). For example, NMP may be produced from organ/structure/tissue/cell cultures (e.g., body fragments, appendages, organs, eggs, exoskeletons, embryos, spores, mycelium, hyphae, thalli, suspension cultures, cell walls, inner or outer membranes, gametophytes, sporophytes, polymerases, glycerol-ether lipids, metabolites, flagella, pili, ribosomes, or organelles) or their offspring. The source may be at any stage of development. In some embodiments, NMP is produced by insects or fungi (e.g., cricket, yeast, or mushrooms). In some embodiments, NMP is produced by bacteria or archaea (e.g., e.coli). In some embodiments, NMP is produced from algae (e.g., kelp or chlorella). In some embodiments, NMP is produced by an animal organ (e.g., brain or blood).

NMP can be produced and purified by a variety of methods, for example, by using a density gradient (iodixanol or sucrose) in combination with ultracentrifugation and/or methods to remove accumulated contaminants (e.g., precipitation or size exclusion chromatography).

In some cases, NMP of the compositions and methods of the present invention can be isolated from arthropods, fungi, archaea or bacteria, or portions thereof, and used without further modification of NMP. In other cases, NMP may be modified prior to use, as further outlined herein. In some cases, the NMP is PMP. In some cases, NMP of the compositions and methods of the invention may be isolated from plants or parts thereof and used without further modification of NMP. In other cases, NMP may be modified prior to use, as further outlined herein.

Lipid reconstitution Natural messenger bag (LNMP)

Lipid reconstituted NMP (LNMP) is used herein. LNMP refers to NMP derived (e.g., enriched, isolated, or purified from) lipid structures (e.g., lipid bilayer, monolayer, multilamellar structures; e.g., vesicle lipid structures) from natural sources, wherein the lipid structures are disrupted (e.g., by lipid extraction disruption) and reassembled or reconstituted in a liquid phase (e.g., a cargo-containing liquid phase) using standard methods, e.g., by methods including lipid membrane hydration and/or solvent injection, to produce LNMP, as described herein. For example, plant LNMP may be referred to as LPMP (lipid reconstituted plant messenger package), which is derived from a plant source.

Methods for lipid reconstitution of NMP (e.g., LPMP) also include sonication, freeze/thaw treatment, and/or lipid extrusion, if desired, e.g., to reduce the size of the reconstituted LNMP. Or a microfluidic device (e.g., nanoAssemblr: IGNITE ^TM microfluidic instrument (Precision NanoSystems)) may be used to generate LNMP (e.g., LPMP).

In some embodiments, the LNMP (e.g., LPMP) is produced by a method comprising (a) providing a plurality of purified NMPs (e.g., purified PMPs), (b) treating the plurality of NMPs (e.g., PMPs) to produce a lipid membrane, (c) reconstituting the lipid membrane in an organic solvent or combination of solvents to produce a lipid solution, and (d) treating the lipid solution of step (c) in a microfluidic device comprising an aqueous phase to produce the LNMP (e.g., LPMP).

In some cases, treating the plurality of NMPs (e.g., PMPs) to produce a lipid membrane includes extracting lipids from the plurality of NMPs, e.g., using the Bligh-Dyer method (Bligh and Dyer, J Biolchem Physiol, 37:911-917, 1959). The extracted lipids may be provided as a stock solution (e.g., a solution in chloroform: methanol). Generating the lipid membrane may include, for example, evaporating the solvent with a stream of an inert gas (e.g., nitrogen).

Natural lipid

LNMP can comprise 10% to 100% lipid of lipid structure derived from natural sources (e.g., lemon or algae), e.g., can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% lipid of lipid structure derived from natural sources. The LNMP can comprise all or part of the lipid material present in the lipid structure from a natural source (e.g., lemon or algae), e.g., it can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the lipid material present in the lipid structure from a natural source. The LNMP may contain no, some, or all proteinaceous matter present in lipid structures from natural sources (e.g., lemon or algae), e.g., may contain 0%, less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, or 100% of proteinaceous matter present in lipid structures from natural sources (e.g., lemon or algae). In some cases, the lipid bilayer of the LNMP does not contain a protein. In some cases, the lipid structure of the LNMP comprises a reduced amount of protein relative to the lipid structure of natural origin.

In some embodiments, the natural lipids of LNMP are extracted from a plant source such as lemon or algae. In some embodiments, the natural lipids of LNMP are extracted from bacterial sources such as Escherichia or Salmonella.

In some embodiments, the natural lipids of LNMP are extracted from lemon or algae.

Exogenous lipids

LNMP can be modified to include a heterologous agent (e.g., a cell penetrating agent) that is capable of increasing cellular uptake (e.g., animal cellular uptake (e.g., mammalian cellular uptake, e.g., human cellular uptake), plant cellular uptake, bacterial cellular uptake, or fungal cellular uptake) relative to unmodified LNMP. For example, the modified LNMP can include (e.g., be loaded with, e.g., encapsulated by, or conjugated to) a cell penetrating agent (e.g., an ionizable lipid), or can be formulated with (e.g., suspended or resuspended in a solution comprising) a cell penetrating agent (e.g., an ionizable lipid). Each modified LNMP can comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

LNMP can include one or more exogenous lipids, such as lipids that are exogenous to the natural source (e.g., derived from a source that does not produce the LNMP or a source portion thereof). The lipid composition of the LNMP can include 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipid. In some examples, the exogenous lipid (e.g., ionizable lipid) is added in an amount of 25% (w/w) or 40% (w/w) of the total lipid in the formulation. In some examples, the exogenous lipid is added to the formulation prior to step (b), e.g., mixed with the extracted NMP lipid prior to step (b).

Exemplary exogenous lipids include ionizable lipids. The ionizable lipids in the LNMP compositions herein include one or more of the group i) -iv) compounds described herein.

Exogenous lipids can also include cationic lipids.

In some cases, the exogenous lipid may also include an ionizable lipid or cationic lipid selected from 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) azadiyl) bis (dodecane-2-ol) (C12-200), DLin-MC3-DMA (MC 3), dioleoyl-3-trimethylammonium propane (DOTAP), DC-cholesterol, DOTAP, ethylpc, GL67, DLin-KC2-DMA (KC 2), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5 (Moderna), cationic sulfonamide amino lipids, amphiphilic zwitterionic amino lipids 、DODAC、DOBAQ、YSK05、DOBAT、DOBAQ、DOPAT、DOMPAQ、DOAAQ、DMAP-BLP、DLinDMA、DODMA、DOTMA、DSDMA、DOSPA、DODAC、DOBAQ、DMRIE、DOTAP- cholesterol, GL67A and 98N12-5, or combinations thereof.

In some embodiments, the exogenous lipid may further comprise an ionizable lipid or cationic lipid selected from the group consisting OF C12-200, MC3, DODAP, DC-cholesterol, DOTAP, ethyl PC, GL67, KC2, MD1, OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5 (Moderna), cationic sulfonamide amino lipids, and amphiphilic zwitterionic amino lipids, or a combination thereof. In some embodiments, the ionizable lipid is selected from the group consisting of C12-200, MC3, DODAP, and DC-cholesterol, or a combination thereof. In some cases, the ionizable lipid is one that is ionizable. In some embodiments, the ionizable lipid is 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) (2-hydroxydodecyl) amino) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol) (C12-200) or (6 z,9z,28z,31 z) -tricyclodeca-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate, DLin-MC3-DMA (MC 3). In some cases, the exogenous lipid is a cationic lipid. In some embodiments, the cationic lipid is DC-cholesterol or dioleoyl-3-trimethylammoniopropane (DOTAP).

In some cases, the LNMP comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

In some cases, the LNMP comprises at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% of the ionizable lipid in a molar ratio, e.g., 1% -10%, 10% -20%, 20% -30%, 30% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80%, or 80% -90% of the ionizable lipid in a molar ratio, e.g., about 30% -75% of the ionizable lipid in a molar ratio (e.g., about 30% -75% of the ionizable lipid in a molar ratio). In some embodiments, the LNMP comprises 25% C12-200. In some embodiments, the LNMP comprises a molar ratio of 35% C12-200. In some embodiments, the LNMP comprises a molar ratio of 50% C12-200. In some embodiments, the LNMP comprises 40% MC3. In some embodiments, the LNMP comprises a molar ratio of 50% C12-200. In some embodiments, the LNMP comprises 20% or 40% DC-cholesterol. In some embodiments, the LNMP comprises 25% or 40% DOTAP.

The agent can increase uptake of the entire LNMP, or can increase uptake of a portion or component of the LNMP (e.g., mRNA or circRNA therapeutic agent carried by the LNMP). The extent of increase in cellular uptake can vary depending on the natural source or source fraction from which the composition is derived, the LNMP formulation, and other modifications made to the LNMP, e.g., cellular uptake (e.g., animal cellular uptake, plant cellular uptake, bacterial cellular uptake, or fungal cellular uptake) of the modified LNMP can be increased by at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the unmodified LNMP. In some cases, the increased cellular uptake is at least 2-fold, 4-fold, 5-fold, 10-fold, 100-fold, or 1000-fold increased cellular uptake relative to unmodified LNMP.

In some embodiments, LNMPs modified with ionizable lipids encapsulate negatively charged polynucleotides more efficiently than LNMPs not modified with ionizable lipids. In some aspects, LNMP modified with an ionizable lipid alters the biodistribution relative to LNMP not modified with an ionizable lipid. In some aspects, the LNMP modified with the ionizable lipid alters (e.g., increases) fusion with endosomal membranes of the target cells relative to LNMP not modified with the ionizable lipid.

Ionizable lipids

In some embodiments, the ionizable lipid has at least one (e.g., one, two, three, four, or all five) of the following listed features:

(i) At least 2 ionizable amines (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, or more than 6 ionizable amines, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ionizable amines);

(ii) At least 3 lipid tails (e.g., at least 3, at least 4, at least 5, at least 6, or more than 6 lipid tails, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 lipid tails), wherein each lipid tail is independently at least 6 carbon atoms in length (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or more than 18 carbon atoms in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 carbon atoms in length);

(iii) An acid dissociation constant (pKa) of about 4.5 to about 7.5 (e.g., a pKa of about 4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4 or 7.5 (e.g., a pKa of about 6.5 to about 7.5 (e.g., a pKa of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5));

(iv) Ionizable amine and heteroorganic groups, and

(V) The ratio of N to P (amine of ionizable lipid: phosphate of mRNA or circRNA) is at least 3 (or at least 4).

In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group. In some embodiments, the heteroorganic group is a hydroxyl group. In some embodiments, the heteroorganic group comprises a hydrogen bond donor. In some embodiments, the heteroorganic group comprises a hydrogen bond acceptor. In some embodiments, the heteroorganic group is-OH, -SH, - (CO) H, -CO ₂H、-NH₂、-CONH₂, optionally substituted C ₁-C₆ alkoxy, or fluorine.

In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

The ionizable lipid in the LNMP composition comprises one of the compounds of groups i) through iv) discussed below.

Ionizable lipid compounds i)

In some embodiments, the ionizable lipid is a compound represented by formula I below, a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

Wherein the method comprises the steps of

Each X is independently a biodegradable moiety, and

W isEither (or) or (b)Wherein:

R ₅ is OH, SH or NR ₁₀R₁₁;

Each s is independently 1, 2, 3, 4, or 5;

Each u is independently 1, 2, 3, 4, or 5;

t is 1, 2, 3, 4 or 5;

Q is O, S or NR ₁₃, where each R ₁₃ is H or C ₁-C₅ alkyl.

In some embodiments, B is C ₃-C₂₀ alkyl.

In some embodiments, W in formula (I) may also beWherein:

Each R ₇ and each R ₈ are independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH, SH, (CH ₂)_vR₁₇ or NR ₁₀R₁₁, wherein each R ₁₀ and R ₁₁ are independently H, C ₁-C₃ alkyl, or R ₁₀ and R ₁₁ together form a heterocycle;

each v is independently 0,1, 2, 3, 4, or 5;

r ₁₇ is OH, SH or N (CH ₃)₂; and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

V is C ₂-C₁₀ alkenylene, C ₂-C₁₀ alkynylene, or C ₂-C₁₀ heteroalkylene;

Each R ₆ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, or cycloalkyl, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

R ₁₄ is a heterocycle;

each v is independently 0,1, 2, 3, 4 or 5, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

Z is O, S, -C ((CH ₂)_vN(R₁₅)₂) -or N (R ₁₅), wherein R ₁₅ is H, C ₁-C₄ branched or unbranched alkyl and v is 0, 1, 2, 3, 4 or 5;

Each R ₁₀ is independently H or C ₁-C₃ alkyl, and

Each u is independently 0,1, 2, 3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

Each Y is a divalent heterocyclic ring;

Q is O, S or NH, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

R ₁₄ is a heterocycle, NR ₁₀R₁₁、C(O)NR₁₀R₁₁, or C (S) NR ₁₀R₁₁, wherein each R ₁₀ and R ₁₁ is independently H, C ₁-C₃ alkyl, C ₃-C₇ cycloalkyl, C ₃-C₇ cycloalkenyl, optionally substituted with one or more NH and/or oxo groups, or R ₁₀ and R ₁₁ together form a heterocycle;

R ₁₆ is H, =o, =s or CN;

each v is independently 0,1, 2, 3, 4 or 5, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

T is-NHC (O) O-, -OC (O) NH-, or optionally surrounded by one or more- (CH ₂)_vOH、-(CH₂)_v SH) and/or a divalent heterocycle substituted by a- (CH ₂)_v -halo group);

R ₁₇ is OH, SH, or N (CH ₃)₂;

each v is independently 0,1, 2, 3, 4 or 5, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, W in formula (I) may also beWherein:

T is-NHC (O) O-, -OC (O) NH-or a divalent heterocycle, and

Each u is independently 1,2,3, 4, or 5.

In some embodiments, when Z is present, adjacent R ₁ and R ₂ cannot be OH, NR ₁₀R₁₁, or SH.

In some embodiments, the heterocycle is piperazine, piperazine dione, piperazine-2, 5-dione, piperidine, pyrrolidine, piperidinol, dioxopiperazine, bipiperazine, aromatic or heteroaromatic.

In some embodiments, the ionizable lipid is a compound represented by formula (IX), a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing:

Wherein

Each X is independently a biodegradable moiety;

Each q is independently 2,3, 4 or 5;

Each R ₆ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl or cycloalkyl;

Each R ₇ and each R ₈ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, halogen, OH, SH, (CH ₂)_vR₁₇ or NR ₁₀R₁₁, wherein each v is independently 0,1, 2,3,4 or 5, and R ₁₇ is OH, SH or N (CH ₃)₂; and

Each m is independently 1,2,3,4, 5, 6, 7, 8, 9, or 10.

In some embodiments, V is a branched or unbranched C ₂-C₃ alkylene group. In some embodiments, V is C ₂-C₃ alkylene substituted with OH. In some embodiments, V is a branched or unbranched C ₂-C₃ alkenylene group. In some embodiments, each R ₆ is independently H or methyl.

In some embodiments, the ionizable lipid is represented by one of the following formulas:

Or (b)

,

Wherein the definition of the variables is the same as that in formula (X).

In some embodiments, the disclosure relates to ionizable lipids of formula (XI), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing:

Wherein

Each X is independently a biodegradable moiety;

Each s is independently 1, 2, 3, 4, or 5;

T is-NHC (O) O-, -OC (O) NH-; or a divalent heterocycle optionally substituted with one or more- (CH ₂)_vOH、-(CH₂)_vSH、-(CH₂)_v -halo) groups;

each v is independently 0,1, 2, 3, 4 or 5, and

Each m is independently 1,2,3,4, 5, 6, 7, 8, 9, or 10.

In some embodiments, T is a divalent heterocycle (e.g., a divalent piperazine or a divalent dioxopiperazine) optionally substituted with- (CH ₂)_v OH), wherein v is independently 0,1, or 2.

In some embodiments, in each of the formulas above, X is -OC(O)-、-C(O)O-、-SS-、-N(R¹⁸)C(O)-、-C(O)N(R¹⁸)-、-C(O-R₁₃)-O-、-C(O)O(CH₂)_a-、-OC(O)(CH₂)_a-、-C(O)N(R¹⁸)(CH₂)_a-、-N(R¹⁸)C(O)(CH₂)_a-、-C(O-R₁₃)-O-(CH₂)_a-, wherein each R ¹⁸ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R ₁₃ is independently C ₃-C₁₀ alkyl, and each a is independently 0-16. In one embodiment, each X is independently-OCO-, -COO-, -NHCO-, or-CONH-. In one embodiment, at least one X is-SS-.

Further embodiments of the ionizable lipids of formula (I) in group I) ionizable lipid compounds can be found in PCT application No. PCT/US22/50725 filed on month 22 of 2022, 11 (the contents of which are incorporated herein by reference in their entirety). In particular, all of the ionizable lipids of formulas (I) - (XII) of PCT application No. PCT/US22/50725 are suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

Certain exemplary ionizable lipid compounds disclosed herein are listed in table I below.

Table i. exemplary ionizable lipid compounds.

Ionizable lipid compound ii)

In some embodiments, the ionizable lipid is a compound represented by formula II below, a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

wherein:

is a cyclic or heterocyclic moiety;

Y is alkyl, hydroxy, hydroxyalkyl or ;

each M is independently a biodegradable moiety;

Each of R ₃₀、R₄₀、R₅₀、R₆₀、R₇₀、R₈₀、R₉₀、R₁₀₀、R₁₁₀ and R ₁₂₀ is independently H, C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally interrupted by a heteroatom or substituted by OH, SH or halogen, or cycloalkyl or substituted cycloalkyl;

each of l and m is an integer of 1 to 10;

t1 is an integer from 0 to 10, and

W is hydroxy, substituted or unsubstituted hydroxyalkyl, substituted or unsubstituted amino, substituted or unsubstituted aminocarbonyl, or substituted or unsubstituted heterocyclyl or heteroaryl.

In some embodiments, Y is hydroxy or。

In some embodiments of the present invention, in some embodiments,Selected from pyrrolidine, piperidine, piperazine, cyclohexane, cyclopentane, tetrahydrofuran, tetrahydropyran, morpholine and dioxane. In some embodiments of the present invention, in some embodiments,Selected from the group consisting of: And 。

In some embodiments, the ionizable lipid is of the formulaAnd (3) representing. All variables in this formula are as defined and exemplified in the embodiments above.

In some embodiments, the ionizable lipid is represented by the formula:

, Or (b) ,

Wherein:

t1 is an integer from 0 to 10;

each M is independently a biodegradable moiety;

Each m1 is independently an integer from 3 to 6,

Each l1 is independently an integer from 4 to 8,

M2 and l2 are each independently an integer from 0 to 3,

R ₈₀ and R ₉₀ are each independently unsubstituted C ₅-C₈ alkyl, or R ₈₀ is H or unsubstituted C ₁-C₄ alkyl, and R ₉₀ is unsubstituted C ₅-C₁₁ alkyl, and

R ₁₁₀ and R ₁₂₀ are each independently unsubstituted C ₅-C₈ alkyl, or R ₁₁₀ is H or unsubstituted C ₁-C₄ alkyl, and R ₁₂₀ is unsubstituted C ₅-C₁₁ alkyl. All other variables in these formulae are as defined and exemplified in the embodiments above. In some embodiments, in these formulas, R ₈₀ is H or unsubstituted C ₁-C₂ alkyl, and R ₉₀ is unsubstituted C ₆-C₁₀ alkyl, and R ₁₁₀ and R ₁₂₀ are each independently unsubstituted C ₅-C₈ alkyl. In some embodiments, R ₈₀、R₉₀、R₁₁₀ and R ₁₂₀ are each independently unsubstituted C ₅-C₈ alkyl.

In some embodiments, in the above formula, A is absent, -O-, -N (R ⁷)-,N(R⁷) C (O) -,

-OC (O) -or-C (O) O-, wherein R ⁶ is independently H, alkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, thiol alkyl or N ⁺(R⁷)₃ -alkylene-Q-, and R ⁷ is H or C ₁-C₃ alkyl.

In some embodiments, in the above formula, t1 is 0, 1, 2, 3, or 4, and t is 0, 1, or 2.

In some embodiments, in the above formula, W is hydroxy, hydroxyalkyl, or one of the following:

And

Wherein:

Each Q is independently absent, -O-, -C (O) -, -C (S) -, -C (O) O-, -C (R ⁷)₂-、-C(O)N(R⁷)-、-C(S)N(R⁷) -or-N (R ⁷) -;

Each R ⁶ is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, amino, aminoalkyl, alkylamino, thiol alkyl, or N ⁺(R⁷)₃ -alkylene-Q-;

Each R ⁸ is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, thiol or thiol alkyl, or two R ⁸ together with the nitrogen atom form a ring;

each q is independently 0,1, 2, 3, 4 or 5, and

Each p is independently 0,1, 2, 3, 4, or 5.

In some embodiments, in the above formula,

X is absent, -O-or-C (O) -;

z is-O- -C (O) O-or-OC (O) -;

M is-OC (O) -or-C (O) O-;

Y or Is the component (A) of OH,

Or (b)

,

Each R ^c is independently H or C ₁-C₃ alkyl;

Each t1 is independently 1,2, 3, or 4;

Each of R ₃₀、R₄₀、R₅₀ and R ₆₀ is independently H or C ₁-C₄ branched or unbranched alkyl;

R ₇₀ is H, and each of R ₈₀ and R ₉₀ is independently H or C ₁-C₁₂ branched or unbranched alkyl;

And each of R ₁₁₀ and R ₁₂₀ is independently H or C ₁-C₁₂ branched or unbranched alkyl, provided that at least one of R ₈₀ and R ₉₀ is not H and at least one of R ₁₁₀ and R ₁₂₀ is not H;

l is 3 to 7, and

M is 1 to 5.

In some embodiments, in the above formula, Y orIs the component (A) of OH,

Or (b)。

Further embodiments of the ionizable lipids of formula (II) in group II) ionizable lipid compounds can be found in PCT application No. PCT/US23/16300 filed on 3/24, 2023, the contents of which are incorporated herein by reference in their entirety. In particular, all ionizable lipids of formula （I）、（IA-1）、（IA-2）、（IIA）-（IIC）、（IIA-1）、（IIIA）-（IIIIE）、（IIIC-1）、（IVA-1）-（IVA-3）、（IVC-1）-（IVC-2）、（VA-1）-（VA-9）、（VC-1）-（VC-6） of PCT application No. PCT/US23/16300 are suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

Certain exemplary ionizable lipid compounds disclosed herein are listed in table II below.

Table II exemplary ionizable lipid compounds

Ionizable lipid compound iii)

In some embodiments, the ionizable lipid is of the formulaA compound represented, a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing, wherein:

R ₂₀ and R ₃₀ are each independently H, C ₁-C₅ branched or unbranched alkyl, or C ₂-C₅ branched or unbranched alkenyl, or

R ₂₀ and R ₃₀ together with the adjacent N atom form a3 to 7 membered ring, optionally substituted by R ^a;

R ₁ and R ₂ together form a ring;

each R ₁₀ and R ₁₁ is independently H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, or

R ₁₀ and R ₁₁ together form a heterocycle;

n is 0, 1, 2, 3 or 4;

Y is O or S;

Each B is independently C ₁-C₁₆ branched or unbranched alkyl, or C ₂-C₁₆ branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms or optionally substituted with OH, SH or halogen;

each X is independently a biodegradable moiety.

In some embodiments, R ₂₀ and R ₃₀ are each independently H or C ₁-C₃ branched or unbranched alkyl. In some embodiments, R ₂₀ and R ₃₀ together with the adjacent N atom form a 3 to 7 membered ring, optionally substituted with R ^a. In some embodiments, R ^a is H, C ₁-C₃ branched or unbranched alkyl or OH. In one embodiment, R ^a is H or OH.

In some embodiments, Z is absent, S, O, or NH. In some embodiments, n is 0,1, or 2.

In some embodiments of the present invention, in some embodiments, the ionizable lipid is a compound represented by formula (V), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

wherein:

R ₁ is H, C ₁-C₃ branched or unbranched alkyl, C ₂-C₃ branched or unbranched alkenyl, OH, halogen, SH or NR ₁₀R₁₁, and

R ₂ is H, OH, halogen, SH or NR ₁₀R₁₁, or

R ₁ and R ₂ together form a ring;

R ₁₀ and R ₁₁ are each independently H or C ₁-C₃ alkyl, or R ₁₀ and R ₁₁ together form a heterocycle;

q is OH or- (OCH ₂CH₂)_uNR₂₀R₃₀),

v is 0, 1, 2, 3 or 4;

y is 0, 1, 2, 3 or 4;

Each a is independently a C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms or optionally substituted with OH, SH or halogen;

Each B is independently a C ₁-C₁₆ branched or unbranched alkyl or a C ₂-C₁₆ branched or unbranched alkenyl group, optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen, and

Each X is independently a biodegradable moiety.

In some embodiments, the disclosure relates to an ionizable lipid of one of the following formulas:

Wherein u is 0, 1, 2, 3,4, 5, 6, 7 or 8;v is 0, 1, 2, 3 or 4 and y is 0, 1, 2, 3 or 4. The other variables are as defined above for formulas III) and V).

In some embodiments, in the above formula, X is -OC(O)-、-C(O)O-、-N(R⁷)C(O)-、-C(O)N(R⁷)-、-C(O-R₁₃)-O-、-C(O)O(CH₂)_s-、-OC(O)(CH₂)_s-、-C(O)N(R⁷)(CH₂)_s-、-N(R⁷)C(O)(CH₂)_s-、-C(O-R₁₃)-O-(CH₂)_s-, wherein each R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl, each R ₁₃ is independently C ₃-C₁₀ alkyl, and each s is independently 0-16. In some embodiments of the present invention, in some embodiments, X is-OC (O) -, -C (O) O-; -C (O) O (CH ₂)_s -or-OC (O) (CH ₂)_s -. In some embodiments, s is 0,1,2, 3, 4, 5,6, 7, 8, 9 or 10.

Further embodiments of the ionizable lipids of formula (III) or (V) in group III) ionizable lipid compounds can be found in PCT application No. PCT/US22/50111, filed 11/16 of 2022, the contents of which are incorporated herein by reference in their entirety. In particular, all of the ionizable lipids of formulas (IO) - (VIIO) and (I) - (VIID) of PCT application No. PCT/US22/50111 are suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

Certain exemplary ionizable lipid compounds disclosed herein are listed in table III below.

Table III exemplary cocoa the lipid compound is ionized.

Ionizable lipid compound iv)

In some embodiments, the ionizable lipid is a lipid comprising at least a head group and at least a tail group of formula (TI) or (TI'), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

Or (b)

Wherein:

E are each independently a biodegradable group;

u1 and u2 are each independently 0,1, 2,3, 4, 5, 6 or 7;

Each R ^t is independently H, C ₁-C₁₆ branched or unbranched alkyl or C ₁-C₁₆ branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH or halogen, or cycloalkyl or substituted cycloalkyl;

Represents a bond linking the tail group and the head group, and

Wherein the pKa of the lipid is from about 4 to about 8.

In some embodiments, E is -OC(O)-、-C(O)O-、-N(R⁷)C(O)-、-C(O)N(R⁷)-、-C(O-R₁₃)-O-、-C(O)O(CH₂)_r-、-C(O)N(R⁷)(CH₂)_r- or-S-or-C (O-R ₁₃)-O-(CH₂)_r -, wherein each R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl; R ₁₃ is branched or unbranched C ₃-C₁₀ alkyl; and R is 1,2, 3, 4, or 5. In some embodiments, each E is independently-OC (O) -, -C (O) O-, -N (R ⁷) C (O) -or-C (O) N (R ⁷) -, wherein R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII):

Wherein u3 and u4 are each independently 0,1, 2, 3 or 4. The definition of the other variables in (TII) is the same as defined in (TI) above.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII):

(e.g. (TIIIa) wherein u3 is 0, 1, 2, 3, 4, 5, 6 or 7, and R ^b is independently in each occurrence H or C ₁-C₄ alkyl. The definition of the other variables in (TIII) is the same as that defined in (TI) above.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIV):

Wherein u3 and u4 are each independently 0, 1, 2, 3 or 4. The definition of the other variables in (TIV) is the same as that defined in (TI) above.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TV): (e.g. ) Wherein u3 is 0, 1,2, 3, 4, 5, 6 or 7;R ⁷ is each independently H or methyl, and R ^b is in each case independently H or C ₁-C₄ alkyl. The definition of the other variables in (TV) is the same as defined in (TI) above.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TII'): (e.g. ) Wherein u3 is 0,1, 2, 3, 4, 5, 6 or 7, and R ^b is independently in each occurrence H or C ₁-C₄ alkyl. The definition of the other variables in (TII ') is the same as that defined in (TI') above.

In some embodiments, the lipid comprises at least one head group and at least one tail group of formula (TIII'): (e.g. ) Wherein u3 is 0, 1, 2,3, 4, 5, 6 or 7;R ⁷ is each independently H or methyl, and R ^b is in each case independently H or C ₁-C₄ alkyl. The definition of the other variables in (TIII ') is the same as that defined in (TI') above.

In some embodiments, the lipid comprises at least one tail group of formulae (TII), (TIII), (TIV), (TV), (TII ') and (TIII'), wherein

Each R ⁷ is independently H or methyl;

R ^b is independently in each occurrence H or C ₁-C₄ alkyl;

u1 and u2 are each independently 0,1, 2,3, 4, 5, 6 or 7;

u3 and u4 are each independently 0,1, 2,3, 4, 5, 6 or 7, and

Wherein the lipid has a pKa of about 4 to about 8.

In some embodiments, the lipid comprises two, three, four, or more tail groups of formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII ') and/or (TIII'), and each tail group may be the same or different.

In some embodiments, in any of formulas (T), (TI), (TII) and (TIII), (TIV), (TV), (TI '), (TII ') and/or (TIII '), above, each R ^a is independently C ₁-C₅ branched or unbranched alkyl, C ₂-C₅ branched or unbranched alkenyl, or C ₂-C₅ branched or unbranched alkynyl. In some embodiments, each R ^a is independently a C ₁-C₃ branched or unbranched alkyl group. In one embodiment, each R ^a is methyl.

In some embodiments, in any of formulas (T), (TI), (TII) and (TIII), (TIV), (TV), (TI '), (TII ') and/or (TIII ') above, u1 is 3, 4 or 5. In some embodiments, in any of formulas (T), (TI), (TII) and (TIII), (TIV), (TV), (TI '), (TII ') and/or (TIII ') above, u2 is 0, 1,2 or 3. In some embodiments, in any of formulas (T), (TI), (TII) and (TIII), (TIV), (TV), (TI '), (TII ') and/or (TIII '), u3 and u4 are each independently 1-7, e.g., u3 and u4 are each independently 1,2, 3 or 4.

In some embodiments, the lipid comprises at least one tail of formula (TIII) wherein each R ^a is methyl, R ^b is independently at each occurrence H, ethyl or butyl, u1 is 3-5, u2 is 0-3, and u3 is 1-7 (e.g., 1-4). In some embodiments, the lipid comprises at least two tails of formula (TIII), wherein the tails of the two formulae (TIII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIII), wherein each tail may be the same or different. In some embodiments, the lipid has four tails of formula (TIII), wherein each tail may be the same or different. In some embodiments, in each tail of formula (TIII), each R ^a is methyl, u1 is 3, u2 is 2, and u3 is 4.

In some embodiments, the lipid comprises at least one tail of formula (TII) wherein each R ^a is methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4. In some embodiments, the lipid has at least two tails of formula (TII), wherein the tails of both formulae (TII) are identical. In some embodiments, the lipid has at least two tails of formula (TII), wherein the tails of the two formulae (TII) are the same or different. In some embodiments, the lipid comprises at least three tails of formula (TII), wherein each tail may be the same or different. In some embodiments, the lipid has four tails of formula (TII), wherein each tail may be the same or different.

In some embodiments, the lipid comprises at least one tail of formula (TIV) wherein each R ^a is methyl, u1 is 3-5, u2 is 0-3, u3 is 1-4, and u4 is 1-4. In some embodiments, the lipid comprises at least two tails of formula (TIV), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TIV), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TIV), wherein each tail may be the same or different.

In some embodiments, the lipid comprises at least two tails of formula (TV), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least three tails of formula (TV), wherein each tail may be the same or different. In some embodiments, the lipid comprises at least four tails of formula (TV), wherein each tail may be the same or different.

In some embodiments, the lipid has at least two tails of formula (TII'), wherein each tail may be the same or different. In some embodiments, the lipid has at least three tails of formula (TII'), wherein each tail may be the same or different. In some embodiments, the lipid has at least four tails of formula (TII'), wherein each tail may be the same or different.

In some embodiments, the lipid has at least two tails of formula (TIII'), wherein each tail may be the same or different. In some embodiments, the lipid has at least three tails of formula (TIII'), wherein each tail may be the same or different. In some embodiments, the lipid has at least four tails of formula (TIII'), wherein each tail may be the same or different.

In some embodiments, the lipid has at least one tail of formula (TII) and/or at least one tail of formula (TIII), the lipid further comprising at least one tail not having formula (T), (TI), (TII), (TIII), (TIV), (TV), (TII ') and/or (TIII'). That is, the lipid also comprises at least one tail that does not comprise a gem-di (gem-di) functional group bonded to the same carbon next to E (e.g., -C (O) O-).

In some embodiments, the lipid further comprises at least one tail not having formula (T), (TI), (TII), (TIV), (TV), (TI '), (TII ') and/or (TII '). That is, the lipid also comprises at least one tail that does not comprise a gem-di-functional group bonded to the same carbon next to E.

In some embodiments, the lipid further comprises at least one tail of formula (TNG-I):

Wherein

Each E is independently a biodegradable group as described herein (e.g., -OC (O) -, -C (O) O-, -N (R ⁷) C (O) -, -S-, or-C (O) N (R ⁷) -;

u1 and u2 are each independently 0,1, 2,3, 4, 5, 6 or 7, and

R ⁷ is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl, or aminoalkyl.

In some embodiments, at least one tail of formula (TNG-I) may be formed fromOr (b)Representation of wherein

U3 and u4 are each independently 0,1, 2,3, 4, 5, 6 or 7, and

R ^b is in each case independently H or C ₁-C₄ alkyl.

All embodiments of the definitions above in relation to E, R ^b、R^t, u1, u2, u3 and u4 (as described above for tail groups of formula (T), (TI), (TII), (TIV), (TV), (TII ') or (TII') comprising a gem-di-functional group bonded to the same carbon beside E) apply to tail groups of formula (TNG-I), (TNG-II) or (TNG-III) not comprising a gem-di-functional group bonded to the same carbon beside E.

In some embodiments, the lipid further comprises at least two tails that do not have formula (T), (TI), (TII), (TIV), (TV), (TI '), (TII ') and/or (TIII '). In some embodiments, the lipid comprises two tail groups of formula (TNG-II) or (TNG-III), wherein each tail group may be the same or different.

In some embodiments, the lipid further comprises at least three tails that do not have formula (T), (TI), (TII), (TIV), (TV), (TI '), (TII ') and/or (TIII '). In some embodiments, the lipid comprises three tail groups of formula (TNG-II) or (TNG-III), wherein each tail group may be the same or different.

In some embodiments, the head group of the lipid HAs the structure of formula (HA-I):

Wherein:

R ₂₀ and R ₃₀ are each independently H, C ₁-C₅ branched or unbranched alkyl or C ₂-C₅ branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms or substituted by OH, SH, halogen or cycloalkyl groups, or

Each of R ₁ and R ₂ is independently H, C ₁-C₃ branched or unbranched alkyl, or C ₂-C₃ branched or unbranched alkenyl, OH, halogen, SH or NR ₁₀R₁₁, or R ₁ and R ₂ together form a ring;

n is 0, 1, 2, 3 or 4;

Z is absent, O, S or NR ₁₂ wherein R ₁₂ is H or C ₁-C₇ branched or unbranched alkyl, provided that when Z is present, adjacent R ₁ and R ₂ cannot be OH, NR ₁₀R₁₁, SH.

In some embodiments, R ₂₀ and R ₃₀ together with the adjacent N atoms form a 3-to 7-membered heterocyclic or heteroaromatic ring containing one or more heteroatoms, optionally substituted with one or more OH, SH, halogen, alkyl or cycloalkyl groups.

In some embodiments, the head group of the ionizable lipid HAs the structure of formula (HA-IA):

wherein:

m is 1, 2, 3, 4, 5, 6, 7 or 8;

n is 0, 1, 2, 3 or 4;

Z is absent, O, S or NR ₁₂ wherein R ₁₂ is H or C ₁-C₇ branched or unbranched alkyl, provided that when Z is present, adjacent R ₁ and R ₂ cannot be OH, NR ₁₀R₁₁, or SH, and

Represents a bond connecting a head group and a tail group.

In some embodiments, the head group of the ionizable lipid HAs the structure of formula (HA-III):

Wherein Z is absent, O, S or NR ₁₂, and R ₁₂ is H or C ₁-C₇ branched or unbranched alkyl. The definition of the other variables in (HA-III) is the same as that defined in (HA-IA) above.

In some embodiments, the head group hasOr (b)Wherein:

rc is H or alkyl, optionally substituted with OH, and

M1 is 1, 2 or 3.

In some embodiments, the head group of the ionizable lipid HAs the structure of formula (HA-V):

wherein:

r ₁ is H, C ₁-C₃ alkyl, OH, halogen, SH, or NR ₁₀R₁₁;

V and y are each independently 1,2,3 or 4.

In some embodiments, the head group of the ionizable lipid HAs the structure of formula (HA-VI):

. All variables in (HA-VI) are as defined above for (HA-V).

In some embodiments, in any of the above formulas (HA-V) or (HA-VI), each R ₂₀ and R ₃₀ is independently C ₁-C₃ alkyl. In one embodiment, each R ₂₀ and R ₃₀ is independently methyl.

In some embodiments, the head group of the ionizable lipid has the structure of formula (HB-I):

Wherein W is

Or (b),

Wherein the method comprises the steps of

R ₅ is OH, SH, (CH ₂)_s OH or NR ₁₀R₁₁;

Each R ₂₀ is independently H or C ₁-C₃ branched or unbranched alkyl;

R ₁₆ is H, =o, =s or CN;

Each of s, u and t is independently 1, 2, 3, 4 or 5;

each v is independently 0,1, 2, 3, 4, or 5;

Each Y is a divalent heterocyclic ring;

Q is O, S, CH ₂ or NR ₁₃, wherein each R ₁₃ is H or C ₁-C₅ alkyl;

T is-NHC (O) O-, -OC (O) NH-or a divalent heterocycle.

In some embodiments, in formula (HB-I), W isWherein:

Each R ₆、R₇ and R ₈ is independently H or methyl, and

Each of u and t is independently 1,2 or 3.

In some embodiments, in formula (HB-I), W isWherein:

R ₁₆ is H or = O;

Each of u and v is independently 1,2 or 3.

In some embodiments, in formula (HB-I), W isWherein:

each R ₆ is independently H or methyl;

each u is independently 1,2 or 3, and

V is C ₂-C₆ alkylene or C ₂-C₆ alkenylene.

In some embodiments, in formula (HB-I), W isWherein:

each R ₆ is independently H or methyl;

Each R ₇ is independently H;

Each R ₈ is methyl;

each u is independently 1,2 or 3, and

V is C ₂-C₆ alkylene or C ₂-C₆ alkenylene.

In some embodiments, in formula (HB-I), W isWherein:

each u is independently 1,2 or 3, and

T is a divalent nitrogen-containing 5-or 6-membered heterocycle.

In some embodiments, in formula (HB-I), W is

Wherein:

each u is independently 1,2 or 3;

Q is O;

Each Z is independently NR ₁₂, and

R ₁₂ is H or C ₁-C₃ alkyl.

In some embodiments, the structure of the head group is:

Or (b) Wherein each of u and t is independently 1 or 2.

In some embodiments, the head group of the ionizable lipid has the structure of formula (HC-I):

Wherein

Is a cyclic or heterocyclic moiety;

y is alkyl, hydroxy, hydroxyalkyl, Or (b);

t is 0,1, 2 or 3;

t1 is an integer from 0 to 10, and

In some embodiments, the head group has the structure of the formula:

Or (b) 。

In some embodiments, in the above formula,

A is absent, -O-, -N (R ⁷) -, -OC (O) -or-C (O) O-;

X is absent, -O-or-C (O) -, and

Z is-O- -C (O) O-or-OC (O) -.

In some embodiments, the head group has the structure of the formula:

Or (b) Wherein t1 is 0, 1,2 or 3.

In some embodiments, W is hydroxy, substituted or unsubstituted hydroxyalkyl, or one of the following moieties:

And ;

Wherein the method comprises the steps of

R ₆ is independently H, alkyl, hydroxy, hydroxyalkyl, alkoxy, -O-alkylene-O-alkyl, -O-alkylene-N (R ⁷)₂, amino, alkylamino, aminoalkyl, thiol alkyl or N ⁺(R⁷)₃ -alkylene-Q-;

Each R ⁸ is independently H, alkyl, hydroxyalkyl, amino, aminoalkyl, alkylamino, thiol or thiol alkyl, heterocyclyl, heteroaryl, or two R ⁸ together with the nitrogen atom may form a ring, optionally substituted with one or more alkyl, hydroxy, hydroxyalkyl, alkoxy, alkylaminoalkyl, alkylamino, aminoalkyl;

q is 0, 1,2, 3, 4 or 5, and

P is 0, 1,2, 3, 4 or 5.

In some embodiments, W is one of the following:

,,,,,

Or (b) 。

In some embodiments, the ionizable lipid is of the formulaA compound represented, a pharmaceutically acceptable salt thereof, and a stereoisomer of any of the foregoing, wherein

R ₁ is each independently H, C ₁-C₃ alkyl, OH, halogen, SH or NR ₁₀R₁₁;R₁ and R ₂ may together form a ring, R ₁₀ and R ₁₁ are each independently H, C ₁-C₃ alkyl, and R ₁₀ and R ₁₁ may together form a heterocyclic ring;

r ₂ is each independently H, C ₁-C₃ alkyl, OH, halogen, SH or NR ₁₀R₁₁;R₁ and R ₂ may together form a ring, R ₁₀ and R ₁₁ are each independently H, C ₁-C₃ alkyl, and R ₁₀ and R ₁₁ may together form a heterocyclic ring;

m is 1, 2, 3, 4, 5, 6, 7 or 8;

n is 0, 1, 2, 3 or 4;

r are each independently 0,1, 2, 3, 4, 5, 6, 7 or 8;

Each R ₃ is independently H or C ₃-C₁₀ alkyl;

R ₄ is each independently H or C ₃-C₁₀ alkyl, provided that at least one of R ₃ and R ₄ is not H;

Z is absent, O, S or NR ₁₂, wherein R ₁₂ is C ₁-C₇ alkyl;

each X is independently X' Or (b)Provided that at least one X in the formula isOr (b)And (C) sum

X' is a biodegradable moiety.

In some embodiments, each X isOr (b). In some embodiments, X' is-OCO-, -COO-, -NR ⁷CO-、-CONR⁷-、-C(O-R₁₃) -O- (acetal )、-COO(CH₂)_s-、-CONH(CH₂)_s-、-C(O-R₁₃)-O-(CH₂)_s-; wherein R ⁷ is H or C ₁-C₃ alkyl; and R ₁₃ is C ₃-C₁₀ alkyl.

In some embodiments, at least one X in the formula isOr (b)Wherein R ⁷ is H or methyl. In one embodiment, each X isOr (b). In one embodiment, each X isOr (b)Wherein R ⁷ is H or methyl.

In some embodiments, m=3. In some embodiments, n=0 or 1. In some embodiments, each RR ₁ and R ₂ is H. In some embodiments, Z is absent.

In some embodiments, Z is S. In some embodiments, Z is O. In some embodiments, Z is NH.

In some embodiments, r is 3. In some embodiments, r is 4.

Further embodiments of the above-described ionizable lipids comprising at least one head group (e.g., a head group of formulae (HA-I), (HA-III), (HA-V), (HA-VI), (HB-I) and (HC-I)) and at least one tail group of formulae (TI) or (T1 ') (e.g., a tail group of formulae (TII), (TIII), TIV, TV, TII ' or TIII ') in group iv ionizable lipid compounds may be found in PCT application number PCT/US23/31669 filed 8-31 in 2023, the contents of which are incorporated herein by reference in their entirety. Furthermore, all ionizable lipids of formulas (LA-I) - (LA-VII), (LB-1) - (LB-VII), (LC-IA) - (LC-IC), (LC-IIA) - (LC-IIC) and (LC-IIIA) - (LC-IIIE) of PCT application No. PCT/US23/31669, filed 8/31 of 2023, are suitable for use as ionizable lipids in the present disclosure, and are incorporated herein by reference in their entirety.

Certain exemplary ionizable lipid compounds disclosed herein are listed in table IV below.

Table iv exemplary ionizable lipid compounds.

In some embodiments, the lipid membrane of the LNMP comprises at least 35% of the lipid compounds of group i), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% of the lipid compounds of group i), e.g., 35% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80% or 80% -90% of the lipid compounds of group i).

In some embodiments, the lipid membrane of the LNMP comprises at least 35% of the lipid compound of group ii), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% of the lipid compound of group ii), e.g., 35% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80% or 80% -90% of the lipid compound of group ii).

In some embodiments, the lipid membrane of the LNMP comprises at least 35% of the lipid compound of group iii), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% of the lipid compound of group iii), e.g., 35% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80% or 80% -90% of the lipid compound of group iii).

In some embodiments, the lipid membrane of the LNMP comprises at least 35% of the lipid compound of group iii), e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% of the lipid compound of group iii), e.g., 35% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80% or 80% -90% of the lipid compound of group iv).

In some cases, the LNMP comprises at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% of the ionizable lipid, e.g., 1% -10%, 10% -20%, 20% -30%, 30% -40%, 40% -50%, 50% -60%, 60% -70%, 70% -80% or 80% -90% of the ionizable lipid, e.g., about 25% -75% of the ionizable lipid (e.g., about 25% -75% of the ionizable lipid), in a molar ratio.

Other ionizable lipids

In LNMP formulations, more than one ionizable lipid may be used for the ionizable lipid component, one or more of the compounds of groups i) -iv) may be used alone or in combination with a different ionizable lipid than the compounds of groups i) -iv).

In some embodiments, the ionizable lipid does not include 1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecyl) amino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5 (Moderna), and 98N12-5.

In some embodiments, the additional ionizable lipid is selected from the group consisting OF 1,1' - ((2- (4- (2- ((2- (bis (2-hydroxydodecylamino) ethyl) piperazin-1-yl) ethyl) azadiyl) bis (dodecane-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5, SM-102 (lipid H), and ALC-315.

In some embodiments, the additional ionizable lipid is represented by formula III below:

wherein R is a C ₈-C₁₄ alkyl group.

The ionizable lipids described herein may include an amine core described herein substituted with one or more (e.g., 1, 2,3,4,5, or 6) lipid tails. In some embodiments, the ionizable lipids described herein include at least 3 lipid tails. The lipid tail may be a C ₈-C₁₈ hydrocarbon (e.g., C ₆-C₁₈ alkyl or C ₆-C₁₈ alkanoyl). The amine core may be substituted at the nitrogen atom with one or more lipid tails (e.g., one hydrogen atom attached to the nitrogen atom may be substituted with a lipid tail).

In some embodiments, the amine core has the structure:

。

in some embodiments, the amine core has the structure:

。

in some embodiments, the amine core has the structure:

。

in some embodiments, the amine core has the structure:

。

in some embodiments, the amine core has the following structure:

。

in some embodiments, the amine core has the structure: 。

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2016/118725 (the entire contents of which are incorporated herein by reference).

In certain embodiments, RNA compositions (e.g., mRNA therapeutic compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2016/118724 (the entire contents of which are incorporated herein by reference).

In certain embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include lipids having the formula 14, 25-ditridecyl 15,18,21,24-tetraaza-trioctadecyl, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publications WO 2013/063268 and WO 2016/205691 (each of which is incorporated herein by reference in its entirety).

In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) and methods of making and using the same comprise a lipid of the formula:

Wherein each instance of R ^L is independently optionally substituted C ₆-C₄₀ alkenyl.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2015/184356 (the entire contents of which are incorporated herein by reference). In some embodiments, the RNA composition (e.g., mRNA composition) and methods of making and using the same include a lipid of the formula:

,

Wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, each R _A is independently hydrogen, optionally substituted C _1-50 alkyl, optionally substituted C _2-50 alkenyl, optionally substituted C _2-50 alkynyl, optionally substituted C _3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C _6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen, and each RB is independently hydrogen, optionally substituted C _1-50 alkyl, optionally substituted C _2-50 alkenyl, optionally substituted C _2-50 alkynyl, optionally substituted C _3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C _6-14 aryl, optionally substituted 5-14 membered heteroaryl, or halogen.

In certain embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include a lipid "Target 23" having a compound structure of the formula:

(Target 23)。

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2016/004202, the entire contents of which are incorporated herein by reference.

In some embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compound, or pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in U.S. provisional patent application serial No. 62/758,179 (incorporated herein by reference in its entirety).

In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) and methods of making and using the same include a lipid having the structure of a compound of the formula:

,

Wherein each R ¹ and R ² is independently H or a C ₁-C₆ aliphatic, each m is independently an integer having a value of 1 to 4, each A is independently a covalent bond or an arylene group, each L ¹ is independently an ester, thioester, disulfide, or anhydride group, each L ² is independently a C ₂-C₁₀ aliphatic, each X ¹ is independently H or OH, and each R ³ is independently a C ₆-C₂₀ aliphatic.

(Compound 1).

(Compound 2).

(Compound 3).

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include lipids as described in j. MCCLELLAN, M.C. King, cell 2010, 141, 210-217, and WHITEHEAD ET al, nature Communications (2014) 5:4277 (the entire contents of which are incorporated herein by reference).

In certain embodiments, the lipid of the RNA composition (e.g., mRNA composition or circRNA composition) and methods of making and using the same include a polypeptide havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2015/199952 (the entire contents of which are incorporated herein by reference).

In some embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2017/004143 (the entire contents of which are incorporated herein by reference).

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2017/075531 (the entire contents of which are incorporated herein by reference).

Wherein one of L ¹ or L ² is -O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x、-S-S-、-C(=O)S-、-SC(=O)-、-NR^aC(=O)-、-C(=O)NR^a-、NR^aC(=O)NR^a-、-OC(=O)NR^a- or-NR ^a C (=O) O-, and the other of L ¹ or L ² is -O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)_x、-S-S-、-C(=O)S-、SC(=O)-、-NR^aC(=O)-、-C(=O)NR^a-、NR^aC(=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-C₁₂ alkylene or C ₁-C₁₂ alkenylene, G ³ is C ₁-C₂₄ alkylene, C ₁-C₂₄ alkenylene, C ₃-C₈ cycloalkylene, and, C ₃-C₈ Cycloalkenylene, R ^a is H or C ₁-C₁₂ alkyl, R ¹ and R ² are each independently C ₆-C₂₄ alkyl or C ₆-C₂₄ alkenyl, R ³ is H, OR ⁵、CN、-C(=O)OR⁴、-OC(=O)R⁴ OR-NR ⁵C(=O)R⁴;R⁴ are C ₁-C₁₂ alkyl, R ⁵ is H OR C ₁-C₆ alkyl, and x is 0, 1 or 2.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2017/117528 (the entire contents of which are incorporated herein by reference). In some embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publication WO 2017/049245 (the entire contents of which are incorporated herein by reference).

In some embodiments, the lipids of the RNA composition (e.g., mRNA composition or circRNA composition) and methods of making and using the same include compounds of the formula:

And And pharmaceutically acceptable salts thereof. For any of these four formulas, R ₄ is independently selected from- (CH ₂)_n Q and- (CH ₂)_n CHQR), Q is selected from the group ：-OR、-OH、-O(CH₂)_nN(R)₂、-OC(O)R、-CX₃、-CN、-N(R)C(O)R、-N(H)C(O)R、-N(R)S(O)₂R、-N(H)S(O)₂R、-N(R)C(O)N(R)₂、-N(H)C(O)N(R)₂、-N(H)C(O)N(H)(R)、-N(R)C(S)N(R)₂、-N(H)C(S)N(R)₂、-N(H)C(S)N(H)(R) and heterocycle consisting of, and n is 1, 2, or 3.

Other suitable lipids for use in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include those described in international patent publications WO 2017/173054 and WO 2015/095340 (each of which is incorporated herein by reference in its entirety). In certain embodiments, RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same include havingLipids of the structure of the compounds, and pharmaceutically acceptable salts thereof.

In some embodiments, the LNMP described herein can include an ionizable lipid as described in the following documents, can be formulated as described in the following documents, or can comprise or be comprised in a composition as described in the following documents ：WO2016118724、WO2016118725、WO2016187531、WO2017176974、WO2018078053、WO2019027999、WO2019036030、WO2019089828、WO2019099501、WO2020072605、WO2020081938、WO2020118041、WO2020146805 or WO2020219876, the entire contents of each of which are incorporated herein by reference.

The ionizable lipids disclosed herein can be used to form LNMP compositions with one or more of the natural lipids disclosed herein. In some embodiments, the LNMP composition is formulated to further comprise one or more therapeutic agents. In some embodiments, the LNMP composition is a lipid nanoparticle encapsulating or associated with the one or more mRNA compositions.

In some embodiments, the RNA composition disclosed herein (e.g., mRNA composition or circRNA composition) has an N/P ratio of at least 3, e.g., an N/P ratio of 3 to 100, 3 to 50, 3 to 30, 3 to 20, 3 to 15, 3 to 12, 3 to 10, 6 to 30, 6 to 20, 6 to 15, or 6 to 12. For example, the N/P ratio may be 6.+ -. 1, or the N/P ratio may be 6.+ -. 0.5. In some embodiments, the N/P ratio is about 6. In some embodiments, the N/P ratio is about 3 (e.g., 3±1 or 3±0.5). In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) has an N/P ratio of about 12 to about 17, e.g., an N/P ratio of about 15±1, or an N/P ratio of about 15±0.5. In some embodiments, the N/P ratio is about 15. In some embodiments, the N/P ratio is about 12 (e.g., 12±1 or 12±0.5).

In some embodiments, the present disclosure relates to a composition comprising (I) one or more compounds selected from the group consisting of ionizable lipids of formulas (I) - (III), pharmaceutically acceptable salts thereof, and stereoisomers of any of the foregoing, and (ii) a lipid component. In some embodiments, the composition comprises 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the one or more compounds.

In some embodiments, the present disclosure relates to a composition comprising (i) one or more lipid nanoparticles and (ii) one or more lipid components.

In some embodiments, the one or more lipid components comprise one or more helper lipids and one or more PEG lipids. In some embodiments, the lipid component comprises one or more helper lipids, one or more PEG lipids, and one or more neutral lipids.

Non-limiting examples of neutral lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), palmitoyl phosphatidylethanolamine (POPE), palmitoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (spp), stearoyl phosphatidylethanolamine (DEPE), and mixtures thereof. Other diacyl phosphatidyl choline and diacyl phosphatidyl ethanolamine phospholipids may also be used. The acyl groups in these lipids may be acyl groups derived from fatty acids having a C ₁₀-C₂₄ carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl or oleoyl.

In some embodiments, the RNA composition (e.g., mRNA composition or circRNA composition) comprises a plant sterol, or a combination of plant sterol and cholesterol. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol (b-sitosterol), stigmasterol (stigmasterol), b-sitostanol (b-sitostanol), campesterol, brassicasterol, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of b-sitosterol, b-sitostanol, campesterol, brassicasterol, compound S-140, compound S-151, compound S-156, compound S-157, compound S-159, compound S-160, compound S-164, compound S-165, compound S-170, compound S-173, compound S-175, and combinations thereof. In some embodiments, the phytosterol is selected from the group consisting of compound S-140, compound S-151, compound S-156, compound S-157, compound S-159, compound S-160, compound S-164, compound S-165, compound S-170, compound S-173, compound S-175, and combinations thereof. In some embodiments, the phytosterol is a combination of compound S-141, compound S-140, compound S-143, and compound S-148. In some embodiments, the phytosterols comprise sitosterol or a salt or ester thereof. In some embodiments, the phytosterols comprise stigmasterols or salts or esters thereof.

Other lipids and other agents

The exogenous lipid can be a cell penetrating agent, can be capable of increasing delivery of the polypeptide to a cell by the LNMP, and/or can be capable of increasing loading (e.g., loading efficiency or loading capacity) of the polypeptide. Other exemplary exogenous lipids include sterols and pegylated lipids.

The LNMP may be modified with other components (e.g., lipids, such as sterols, such as cholesterol; or small molecules) to further alter the functional and structural characteristics of the LNMP. For example, the LNMP can be further modified with a stability molecule that increases the stability of the LNMP (e.g., stable for at least one day at room temperature and/or stable for at least one week at 4 ℃).

In some embodiments, the LNMP is modified with a sterol, such as sitosterol, sitostanol, β -sitosterol, 7α -hydroxycholesterol, pregnenolone, cholesterol (e.g., sheep cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or an analog of any sterol (e.g., glycoside, ester, or peptide). In some examples, the exogenous sterol is added to the formulation prior to step (b), for example, by mixing with the extracted NMP lipid prior to step (b). Exogenous sterols may be added in amounts of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more than 90% (w/w) of the total lipids and sterols in the formulation.

In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the LNMP comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or more than 60% sterols (e.g., cholesterol or sitosterol) in a molar ratio, such as 1% -10%, 10% -20%, 20% -30%, 30% -40%, 40% -50% or 50% -60% sterols. In some embodiments LPMP comprises about 35% -50% sterols (e.g., cholesterol or sitosterol) in a molar ratio, such as about 36%, 38.5%, 42.5%, or 46.5% sterols. In some embodiments, the LNMP comprises about 20% to 40% sterols by mole.

In some embodiments, the stability of the LNMP that has been modified with a sterol is altered (e.g., increased) relative to the LNMP that has not been modified with a sterol. In some aspects, the LNMP that has been modified with a sterol has a higher rate of fusion with the membrane of the target cell relative to LNMP that has not been modified with a sterol.

In some cases, the LNMP comprises an exogenous lipid and an exogenous sterol.

In some embodiments, the LNMP is modified with a pegylated lipid. Polyethylene glycol (PEG) may be 1 kDa to 10 kDa in length, and in some aspects PEG of length 2 kDa is used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some cases, the LNMP comprises at least 0.1%、0.2%、0.3%、0.4%、0.5%、0.6%、0.7%、0.8%、0.9%、1%、1.1%、1.2%、1.3%、1.4%、1.5%、1.6%、1.7%、1.8%、1.9%、2%、2.1%、2.2%、2.3%、2.4%、2.5%、2.6%、2.7%、2.8%、2.9%、3%、3.5%、4%、4.5%、5%、10%、20%、30%、40%、50% or more than 50% of the pegylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2 k) by mole ratio, such as 0.1% -0.5%, 0.5% -1%, 1% -1.5%, 1.5% -2.5%, 2.5% -3.5%, 3.5% -5%, 5% -10%, 10% -20%, 20% -30%, 30% -40%, or 30% -50% of the pegylated lipid. In some embodiments, the LNMP comprises a molar ratio of about 0.1% -10% of the pegylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2 k), such as about 1% -3% of the pegylated lipid, e.g., about 1.5% or about 2.5% of the pegylated lipid. In some embodiments, the stability of the LNMP that has been modified with the pegylated lipid is altered (e.g., increased) relative to the LNMP that has not been modified with the pegylated lipid. In some embodiments, the particle size of LNMP that has been modified with a pegylated lipid is altered relative to LNMP that has not been modified with a pegylated lipid. In some embodiments, LNMP that has been modified with a pegylated lipid is less likely to be phagocytosed than LNMP that has not been modified with a pegylated lipid. The addition of pegylated lipids may also affect stability in the gastrointestinal tract and enhance particle migration through mucus. PEG can be used as a method of attaching the targeting moiety.

In some embodiments, the LNMP is modified with an ionizable lipid (e.g., C12-200 or MC 3) and one or both of a sterol (e.g., cholesterol or sitosterol) and a PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2 k).

In some embodiments, the modified LNMP comprises about 5% -50% LNMP lipid (e.g., about 10% -20% LNMP lipid, e.g., about 10%, 12.5%, 16% or 20% LNMP lipid), about 30% -75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 35% -50% sterol (e.g., about 36%, 38.5%, 42.5% or 46.5% sterol), and about 0.1% -10% PEGylated lipid (e.g., about 1% -3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid).

In some embodiments, the modified LNMP comprises about 5% -60% LNMP lipid (e.g., about 10% -20%, 20% -30%, 30% -40%, 40% -50% or 50% -60% LNMP lipid, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50% or 60% LNMP lipid), about 25% -75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 10% -50% sterol (e.g., about 10%, 12.5%, 14%, 16%, 18%, 20%, 36%, 38.5%, 42.5% or 46.5% sterol), and about 0.1% -10% PEGylated lipid (e.g., about 0.5% -5% PEGylated lipid, e.g., about 1% -3% PEGylated lipid or about 1.5% or about 2.5% PEGylated lipid).

In some embodiments, the ionizable lipid, LNMP lipid, sterol, and pegylated lipid comprise about 25% -75%, about 20% -60%, about 10% -45%, and about 0.5% -5%, respectively, of the lipids in the modified NMP.

In some embodiments, the ionizable lipid, natural lipid, sterol, and PEGylated lipid comprise about 30% -75%, about 20% -50%, about 10% -45%, and about 1% -5%, respectively, of the lipids in the modified NMP.

In some embodiments, the ionizable lipid, natural lipid, sterol, and PEGylated lipid comprise about 35% -75%, about 20% -50%, about 10% -45%, and about 1% -5%, respectively, of the lipids in the modified NMP.

In some embodiments, the ionizable lipid, natural lipid, sterol, and pegylated lipid are formulated in a molar ratio of about 35:50:12.5:2.5.

In some embodiments, the ionizable lipid, natural lipid, sterol, and pegylated lipid are formulated in a molar ratio of about 35:50:11.5:3.5.

In some embodiments, the ionizable lipid, natural lipid, sterol, and pegylated lipid are formulated in a molar ratio of about 35:20:42.5:2.5.

In some embodiments, LNMPs that have been modified with ionizable lipids (and/or cationic lipids) and sterols and/or pegylated lipids encapsulate negatively charged cargo (e.g., nucleic acid) more efficiently than LNMPs that have not been modified with ionizable lipids (and/or cationic lipids) and sterols and/or pegylated lipids. The modified LNMP can have an encapsulation efficiency of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more than 99% for cargo (e.g., nucleic acid, e.g., RNA or DNA), e.g., the encapsulation efficiency can be 5% -30%, 30% -50%, 50% -70%, 70% -80%, 80% -90%, 90% -95% or 95% -100%.

Cellular uptake of modified LNMP can be measured by a variety of methods known in the art. For example, LNMP or a component thereof can be labeled with a marker (e.g., a fluorescent marker) that can be detected in isolated cells to confirm uptake.

In some embodiments, the LNMP formulations provided herein comprise two or more different modified LNMPs, e.g., comprise modified LNMPs derived from different unmodified LNMPs (e.g., unmodified LNMPs from two or more different natural sources) and/or comprise modified LNMPs comprising different substances and/or different ratios of ionizable lipids, sterols, and/or pegylated lipids.

In some cases, the organic solvent that dissolves the lipid membrane is dimethylformamide: methanol (DMF: meOH). Or the organic solvent or combination of solvents may be, for example, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethylsulfoxide, acetonitrile, ethanol, acetonitrile, methanol, acetone, methanol, methyl tert-butyl ether, propanol, tetrahydrofuran, dimethylsulfoxide, methanol or dimethylformamide, methanol.

The aqueous phase may be any suitable solution, such as a citrate buffer (e.g., a citrate buffer having a pH of about 3.2), water, or Phosphate Buffered Saline (PBS). The aqueous phase may also comprise a nucleic acid (e.g., siRNA or siRNA precursor (e.g., dsRNA), miRNA or miRNA precursor, mRNA, circRNA or plasmid (pDNA)) or a small molecule.

The lipid solution and the aqueous phase may be mixed in any suitable ratio in the microfluidic device. In some examples, the aqueous phase and the lipid solution are mixed in a 3:1 volume ratio.

The LNMP can optionally include additional agents, such as cell penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. LNMP can carry or be associated with additional agents in a variety of ways to enable delivery of the agent to a target plant or animal, for example, by encapsulation of the agent, incorporation of the agent into the lipid bilayer structure, or association of the agent with the surface of the lipid bilayer structure (e.g., by conjugation). The nucleic acid molecule can be incorporated into the LNMP in vivo or in vitro (e.g., in tissue culture, in cell culture, or synthetically).

Zeta potential

LNMP comprising an ionizable lipid and optionally a cationic lipid (e.g., DC-cholesterol or DOTAP) may have a zeta potential of, for example, greater than-30 mV in the absence of cargo, greater than-20 mV, greater than-5 mV, greater than 0 mV, or about 30 mv in the absence of cargo. In some examples, the LNMP has a negative zeta potential in the absence of cargo, e.g., a zeta potential of less than 0 mV, less than-10 mV, less than-20 mV, less than-30 mV, less than-40 mV, or less than-50 mV. In some examples, the LNMP has a positive zeta potential in the absence of cargo, e.g., a zeta potential of greater than 0 mV, greater than 10 mV, greater than 20 mV, greater than 30 mV, greater than 40 mV, or greater than 50 mV. In some examples, the LNMP has a zeta potential of about 0.

The zeta potential of the LNMP can be measured using any method known in the art. Zeta potential is typically measured indirectly, e.g., calculated using a theoretical model from data obtained using methods and techniques known in the art (e.g., electrophoretic mobility or dynamic electrophoretic mobility). Electrophoretic mobility is typically measured using micro-electrophoresis, electrophoretic light scattering, or tunable resistive pulse sensing. Electrophoretic light scattering is based on dynamic light scattering. In general, zeta potential can be obtained by Dynamic Light Scattering (DLS) measurements, also known as photon correlation spectroscopy or quasi-elastic light scattering.

Plant EV markers

LNMP in RNA compositions (e.g., mRNA compositions or circRNA compositions) and methods of making and using the same can have a range of markers that identify LNMP (e.g., LPMP) as produced using a plant EV and/or including segments, portions, or extracts thereof. As used herein, the term "plant EV marker" refers to a component, such as a plant protein, plant nucleic acid, plant small molecule, plant lipid, or combination thereof, that is naturally associated with a plant and is incorporated into or onto a plant EV in a plant. Examples of plant EV markers can be found, for example, in Rutter and Innes,Plant Physiol. 173(1): 728-741, 2017;Raimondo et al.,Oncotarget. 6(23): 19514, 2015;Ju et al.,Mol. Therapy.21(7):1345-1357, 2013;Wanget al.,Molecular Therapy. 22(3): 522-534, 2014; and REGENTE ET AL, J of exp. Biol. 68 (20): 5485-5496, 2017, each of which is incorporated herein by reference.

Other examples of suitable plant EV markers include those described and listed in International patent application publication No. WO 2021/04301 (the entire contents of which are incorporated herein by reference).

Bacterial EV markers

Bacterial components (e.g., bacterial lipids) in a bacterial-derived lipid composition and methods of making and using the same can have a range of markers that identify the production of bacterial components. As used herein, the term "bacterial EV marker" refers to a component, such as a bacterial protein, bacterial nucleic acid, bacterial small molecule, bacterial lipid, or a combination thereof, that is naturally associated with a bacterium and incorporated into or onto a bacterial EV.

EV markers of natural origin

NMP may have a range of markers that identify NMP as being produced by a particular source EV and/or including segments, portions or extracts thereof. As used herein, the term "EV marker" refers to a component, such as a protein, nucleic acid, small molecule, lipid, or combination thereof, that is naturally associated with a particular source and is incorporated in or on an EV in vivo. Examples of source EV markers include, but are not limited to, peptidoglycan, lipopolysaccharide, ester-linked lipid, ether-linked lipid, circular DNA, chitin, beta-glucan, petunidin (pekilo), mycoprotein, keratin (cerato-platanin), exotoxin, diacylglycerol, triglyceride, phosphatidylcholine, phosphatidylinositol, ornithine lipid, glycolipid, sphingolipid, hopane, or ergosterol.

The source EV marker may comprise a lipid. Examples of lipid markers that may be found in NMP include lipid a, lipopolysaccharide, ergosterol, ornithine Lipid (OL), sulfanyl, diacylglycerol-N, N-trimethylhomoserine (DGTS), glycolipid (GL), diacylglycerol (DAG), hopane (HOP), glucosylceramide, sterol glycoside, ether linked lipid, or combinations thereof.

Other EV markers may include lipids that accumulate in sources in response to abiotic or biotic stressors.

Or the source EV marker may comprise a protein. In some cases, the protein EV marker may be an antimicrobial or antiviral protein naturally occurring from a source, including proteins secreted in response to an abiotic or biotic stressor. Some examples of protein EV markers include, but are not limited to, cecropin (cecropin), mo Lixin (moricin), defensins, peptides enriched in proline and glycine, fungal immunomodulatory proteins, flagellins, encapsulation proteins (encapulins), streptavidin, internalization proteins, pilins, halophiles, or archaea (archaeocin). In some cases, the EV marker may include a protein involved in lipid metabolism. In some cases, the protein EV marker is a cell transport protein in the source. In some cases where the EV marker is a protein, the protein marker may lack a signal peptide normally associated with a secreted protein. Unconventional secreted proteins appear to have several common features, such as (i) lack of leader sequences, (ii) absence of PTM specific for ER or Golgi apparatus, and/or (iii) the influence of brefeldin a, the secretion of which does not block the classical ER/Golgi-dependent secretory pathway. Those skilled in the art can use a variety of publicly available tools for evaluating the signal sequence of a protein or its absence.

Where the EV marker is a protein, the protein may have an amino acid sequence that has at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a known EV marker.

In some cases, EV markers include nucleic acids encoded in the source, such as arthropod, plant, fungus, archaea, or bacterial RNA, DNA, or PNA. For example, NMP may include dsRNA, mRNA, circular RNA (circRNA), viral RNA, microRNA (miRNA), or small interfering RNA (siRNA) encoded by a source. In some cases, the nucleic acid may be a nucleic acid associated with a protein that facilitates long distance transport of RNA. In some cases, the nucleic acid EV marker may be a nucleic acid EV marker involved in host-induced gene silencing (HIGS), HIGS being a process from which the pathogen foreign transcript is silenced. In some cases, the nucleic acid can be microRNA.

Where the EV marker is a nucleic acid, the nucleic acid may have a nucleotide sequence that has at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to a known EV marker.

In some cases, the EV marker includes a compound produced by a source. For example, the compound may be a component of the cell wall (e.g., lipopolysaccharide). For example, the compound may be a defensive compound produced in response to an abiotic or biotic stressor (e.g., a pathogen or extreme environmental stress).

In some cases, NMP may also be identified as being produced by a source EV based on the lack of certain markers (e.g., lipids, polypeptides, or polynucleotides) that are not normally produced by these sources but are normally associated with other organisms (e.g., markers of animal EVs or plant EVs). For example, in some cases, NMP lacks lipids commonly found in animal EVs or plant EVs.

EV markers can be identified using any method known in the art that is capable of identifying small molecules (e.g., mass spectrometry), lipids (e.g., mass spectrometry), proteins (e.g., mass spectrometry, immunoblotting), or nucleic acids (e.g., PCR analysis). In some cases, the NMP compositions described herein include a detectable amount (e.g., a predetermined threshold amount) of the EV markers described herein.

Loading of agents (e.g., nucleic acids)

LNMP is modified to include a therapeutic agent (e.g., a nucleic acid molecule) to form an RNA composition (e.g., an mRNA composition or a circRNA composition). LNMP can carry or be associated with these agents in a variety of ways to enable delivery of the agent to a target organism (e.g., target animal), for example, by incorporating the component into a lipid bilayer structure through an encapsulating agent, or associating the component with the surface of the lipid bilayer structure of LNMP (e.g., by conjugation). In some cases, the agent is included in an LNMP formulation, as described herein.

The agent may be incorporated into or loaded onto the LNMP by any method known in the art that allows for direct or indirect association of the LNMP with the agent. The agent may be incorporated into the LNMP by in vivo methods (e.g., in plants (in planta), e.g., by producing the LNMP from transgenic plants comprising the agent) or in vitro methods (e.g., in tissue culture, or in cell culture), or both in vivo and in vitro methods.

In some cases, the LNMP is loaded in vitro. The substance may be loaded onto or into the LNMP (e.g., may be encapsulated by the LNMP) using, but not limited to, physical, chemical, and/or biological methods (e.g., in tissue culture or in cell culture). For example, the agent may be introduced into the LNMP by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction, or extrusion. In some cases, the agent is incorporated into the LNMP using a microfluidic device, for example, using a method that includes providing LNMP lipids in an organic phase, providing a heterologous functional agent in an aqueous phase, and combining the organic phase and the aqueous phase in the microfluidic device to produce the LNMP that includes the heterologous functional agent. The loaded LNMP can be evaluated to confirm the presence or level of the loaded agent using a variety of methods, such as HPLC (e.g., to evaluate small molecules), immunoblotting (e.g., to evaluate proteins), and/or quantitative PCR (e.g., to evaluate nucleotides). However, those skilled in the art will appreciate that loading the substance of interest into the LNMP is not limited to the methods described above.

In some cases, the agent can be conjugated to the LNMP, wherein the agent is indirectly or directly linked or linked to the LNMP. For example, one or more agents can be chemically linked to the LNMP such that the one or more agents are directly linked (e.g., by covalent or ionic bonds) to the lipid bilayer of the LNMP. In some cases, conjugation of the various agents to the LNMP can be achieved by first mixing one or more agents with a suitable crosslinking agent (e.g., N-ethyl carbodiimide ("EDC"), which is typically used as a carboxyl activator for amide bonding with primary amines and also reacts with phosphate groups) in a suitable solvent. After an incubation period sufficient to allow the agent to attach to the crosslinker, the crosslinker/agent mixture can then be combined with the LNMP, and after another incubation period, subjected to a sucrose gradient (e.g., 8%, 30%, 45%, and 60% sucrose gradient) to separate the free agent and free LNMP from the agent conjugated to the LNMP. As part of combining the mixture with the sucrose gradient and the concomitant centrifugation step, the LNMP conjugated with the agent is then considered as a band in the sucrose gradient, so that the conjugated LNMP can then be collected, washed, and dissolved in a suitable solution for use as described herein.

In some cases, LNMP is associated with agent stabilization before and after delivery of the LNMP to, for example, an animal. In other cases, the LNMP is associated with an agent such that after the LNMP is delivered to, for example, an animal, the agent dissociates from the LNMP.

LNMP can be loaded with various concentrations of agent or can be formulated with various concentrations of agent, depending on the particular agent or use. For example, in some cases, the LNMP is loaded or formulated such that the LNMP formulations disclosed herein include about 0.001, 0.01, 0.1, 1.0, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 95 (or any range of about 0.001 to 95) or higher wt% agent. In some cases, the LNMP is loaded or formulated such that the LNMP formulation includes about 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.1, 0.01, 0.001 (or any range of about 95 to 0.001) or less wt% of the agent. For example, the LNMP formulation can include about 0.001 to about 0.01 wt%, about 0.01 to about 0.1 wt%, about 0.1 to about 1 wt%, about 1 to about 5 wt%, or about 5 to about 10 wt%, about 10 to about 20 wt% of the agent. In some cases, the LNMP may be loaded with an agent or formulated with an agent of about 1, 5, 10, 50, 100, 200, or 500, 1,000, 2,000 (any range of about 1 to 2,000), or higher μg/mL. The LNMP of the invention can be loaded with an agent or the LNMP can be formulated with an agent of about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range of about 2,000 to 1) or less μg/mL.

In some cases, the LNMP is loaded or formulated such that the LNMP formulations disclosed herein include at least 0.001 wt%, at least 0.01 wt%, at least 0.1 wt%, at least 1.0 wt%, at least 2 wt%, at least 3 wt%, at least 4wt%, at least 5 wt%, at least 6 wt%, at least 7 wt%, at least 8 wt%, at least 9 wt%, at least 10 wt%, at least 15 wt%, at least 20 wt%, at least 30 wt%, at least 40 wt%, at least 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, or at least 95 wt% of the agent. In some cases, LNMP may be loaded with or formulated with an agent of at least 1 μg/mL, at least 5 μg/mL, at least 10 μg/mL, at least 50 μg/mL, at least 100 μg/mL, at least 200 μg/mL, at least 500 μg/mL, at least 1,000 μg/mL, at least 2,000 μg/mL.

In some cases, LNMP is formulated with a formulation by suspending the LNMP in a solution comprising or consisting of the formulation, for example by vigorously mixing to suspend or re-suspend the LNMP. The agent (e.g., a cell penetrating agent, such as a nucleic acid, enzyme, detergent, ionic liquid, fluorogenic liquid, or zwitterionic liquid, or ionizable lipid) may comprise, for example, less than 1% or at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the solution.

Pharmaceutical preparation

The modified LNMP is formulated into a pharmaceutical composition (i.e., an RNA composition, such as an mRNA composition or a circRNA composition), for example, for administration to an animal (e.g., a human). The pharmaceutical compositions may be administered to an animal (e.g., human) with pharmaceutically acceptable diluents, carriers and/or excipients. Depending on the mode of administration and the dosage, the pharmaceutical compositions of the methods described herein will be formulated into suitable pharmaceutical compositions to allow for easy delivery. The single dose may be in the desired unit dosage form.

LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) can be formulated for, for example, oral administration, intravenous administration (e.g., injection or infusion), intramuscular or subcutaneous administration to an animal. For injectable formulations, a variety of effective drug carriers are known in the art (see, e.g., remington: THE SCIENCE AND PRACTICE of Pharmacy, 22 nd edition, (2012) and ASHP Handbook on Injectable Drugs, 18 th edition, (2014)).

Suitable pharmaceutically acceptable carriers and excipients are non-toxic to the recipient at the dosages and concentrations employed. Acceptable carriers and excipients can include buffers such as phosphate, citrate, HEPES and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexahydrocarbon quaternary ammonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine and lysine, and carbohydrates such as glucose, mannose, sucrose and sorbitol. LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) can be formulated according to conventional pharmaceutical practice. The concentration of the compound in the formulation will vary depending on a variety of factors, including the dose of the active agent (e.g., LNMP and nucleic acid) to be administered and the route of administration.

For oral administration to animals, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can be prepared in the form of an oral formulation. Formulations for oral use may include tablets, caplets, capsules, syrups, or oral liquid dosage forms containing the active ingredient in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches (including potato starch), calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives (including microcrystalline cellulose), starches (including potato starch), croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, dextrose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, polyvinylpyrrolidone, or polyethylene glycol), and lubricants, glidants, and anti-adherents (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients may be coloring agents, flavoring agents, plasticizers, humectants, buffers, and the like. Formulations for oral use may also be presented as chewable tablets, non-chewable tablets, caplets, capsules (e.g., as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium) in unit dosage form. The compositions disclosed herein may further comprise an immediate release, an extended release or a delayed release formulation.

For parenteral administration to animals, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can be formulated in the form of a liquid solution or suspension and administered by a parenteral route (e.g., subcutaneously, intravenously, or intramuscularly). The pharmaceutical composition may be formulated for injection or infusion. Pharmaceutical compositions for parenteral administration may be formulated using sterile solutions or any pharmaceutically acceptable liquids as excipients. Pharmaceutically acceptable excipients include, but are not limited to, sterile water, physiological saline, or cell culture media (e.g., dulbecco's Modified Eagle Medium, DMEM), alpha modified Eagle Medium (alpha-MEM), and F-12 Medium). Methods of formulation are known in the art, see, e.g., gibson (ed.) Pharmaceutical Preformulation and Formulation (2 nd edition) Taylor & Francis Group, CRC Press (2009).

Polynucleotide

LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) include one or more nucleic acid molecules, e.g., polynucleotides, that encode one or more wild-type or engineered proteins, peptides, or polypeptides. Exemplary polynucleotides (e.g., polynucleotide constructs) include RNA polynucleotides encoding an antigen, such as mRNA, linear polyribonucleotides, or circRNA.

Examples of polypeptides that may be used herein may include enzymes (e.g., metabolic recombinases, helicases, integrases, rnases, dnases, or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas systems, TALENs, or zinc fingers), riboproteins, protein aptamers, or chaperonins.

Polypeptides included herein may include naturally occurring polypeptides or recombinantly produced variants. In some cases, the polypeptide may be a functional fragment or variant thereof (e.g., an enzymatically active fragment or variant thereof). For example, a polypeptide may be a functionally active variant of any of the polypeptides described herein, which has at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98% or 99% identity, e.g., over a specified region or the entire sequence, to a polypeptide described herein or to a sequence of a naturally occurring polypeptide. In some cases, the polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99% or more) identity with the protein of interest.

The polypeptide may be about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, the polypeptide can be less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids, less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less.

The LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can include any number or type (e.g., species) of polypeptides, such as at least about any of 1 polypeptide, 2, 3, 4, 5, 10, 15, 20, or more polypeptides. The appropriate concentration of each polypeptide in the LNMP/RNA composition depends on factors such as efficacy, stability of the polypeptide, the number of different polypeptides in the formulation, and the method of application of the formulation. In some cases, each polypeptide in the liquid formulation is from about 0.1 ng/mL to about 100 mg/mL. In some cases, each polypeptide in the solid formulation is from about 0.1 ng/g to about 100 mg/g.

Nucleic acid encoding a peptide

In some cases, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) includes a heterologous nucleic acid encoding a polypeptide. The length of the nucleic acid encoding the polypeptide may be from about 10 to about 50,000 nucleotides (nt), from about 25 to about 100 nt, from about 50 to about 150 nt, from about 100 to about 200 nt, from about 150 to about 250 nt, from about 200 to about 300 nt, from about 250 to about 350 nt, from about 300 to about 500 nt, from about 10 to about 1000 nt, from about 50 to about 1000 nt, from about 100 to about 1000 nt, from about 1000 to about 2000 nt, from about 2000 to about 3000 nt, from about 3000 to about 4000 nt, from about 4000 to about 5000 nt, from about 5000 to about 6000 nt, from about 6000 to about 7000 nt, from about 7000 to about 8000 nt, from about 8000 to about 9000 nt, from about 9000 to about 10,000 nt, from about 10,000 to about 15,000 nt, from about 10,000 to about 20,000 nt, from about 10,000 to about 25,000 nt, from about 10,000 to about 30,000 nt, from about 10,000 to about 40,000 nt, from about 45,000 nt, from about 50 to about 50,000 nt, or any range therebetween.

LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) can also include active variants of the nucleic acid sequence of interest. In some cases, variants of a nucleic acid have at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98% or 99% identity to the sequence of the nucleic acid of interest, e.g., over a designated region or the entire sequence. In some cases, the invention includes an active polypeptide encoded by a nucleic acid variant described herein. In some cases, the active polypeptide encoded by the nucleic acid variant has at least 70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98% or 99% identity, e.g., over a designated region or the entire amino acid sequence, to the sequence of the polypeptide of interest or to a naturally-derived polypeptide sequence.

Certain methods for expressing nucleic acids encoding proteins may involve expression under the control of a suitable promoter in cells, including insects, yeast, plants, bacteria, or other cells. Expression vectors may include non-transcribed elements such as origins of replication, suitable promoters and enhancers and other 5 'or 3' flanking non-transcribed sequences, as well as 5 'or 3' non-translated sequences such as the necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as SV40 origin, early promoters, enhancers, splicing and polyadenylation sites, may be used to provide other genetic elements necessary for expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

Genetic modifications using recombinant methods are generally known in the art. The nucleic acid sequence encoding the desired gene may be obtained using recombinant methods known in the art, for example, using standard techniques, by screening libraries from cells expressing the gene, by deriving the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene. Alternatively, the gene of interest may be produced synthetically, rather than by cloning.

Expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid encoding a gene of interest to a promoter and incorporating the construct into an expression vector. Expression vectors may be suitable for replication and expression in bacteria. Expression vectors may also be suitable for replication and integration in eukaryotic organisms. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters for expression of the desired nucleic acid sequences.

Additional promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these promoter elements are located in the region 30-110 base pairs (bp) upstream of the start site, but many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible such that promoter function is preserved when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements may be increased to 50 bp a apart before the activity begins to decrease. Depending on the promoter, it appears that the individual elements may act synergistically or independently to activate transcription.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein-Barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

Alternatively, the promoter may be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked thereto when such expression is desired or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The expression vector to be introduced may also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expression cells from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable markers may be carried on separate DNA fragments and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

Reporter genes can be used to identify potentially transformed cells and to assess the function of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient source and encodes a polypeptide that embodies its expression by some readily detectable property (e.g., enzymatic activity). The expression of the reporter gene is analyzed at a suitable time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., ui-Tei et al, FEBS Letters 479:79-82, 2000). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Typically, constructs with minimal 5' flanking regions that show the highest expression levels of the reporter gene are identified as promoters. Such promoter regions may be linked to a reporter gene and used to assess the ability of an agent to regulate promoter-driven transcription.

In some cases, the organism may be genetically modified to alter the expression of one or more proteins. Expression of one or more proteins may be modified at a particular time (e.g., during a developmental or differentiation stage of an organism). In one instance, a composition is provided that alters the expression of one or more proteins, such as proteins that affect activity, structure, or function. Expression of one or more proteins may be limited to a specific location or widely distributed throughout an organism.

mRNA

The LNMP/mRNA composition can include an mRNA molecule, e.g., an mRNA molecule encoding a polypeptide. mRNA molecules may be synthetic and modified (e.g., chemically). mRNA molecules can be chemically synthesized or transcribed in vitro. The mRNA molecule may be placed on a plasmid, such as a viral vector, bacterial vector, or eukaryotic expression vector. In some examples, the mRNA molecules can be delivered to the cells by transfection, electroporation, or transduction (e.g., adenovirus or lentivirus transduction).

In some cases, the modified RNA agents of interest described herein have modified nucleosides or nucleotides. Such modifications are known and described, for example, in WO 2012/019168. Additional modifications are described, for example, in WO 2015/038892, WO 2015/089511, WO 2015/196130, WO 2015/196118 and WO 2015/196128 A2, the entire contents of which are incorporated herein by reference.

In some cases, the modified RNA encoding the polypeptide of interest has one or more terminal modifications, such as a 5' cap structure and/or a poly-a tail (e.g., 100-200 nucleotides in length). The 5 'cap structure may be selected from the group consisting of CapO, capl, ARCA, inosine, nl-methyl-guanosine, 2' fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the modified RNA further comprises a 5' utr and a 3' utr, the 5' utr comprising at least one Kozak sequence. Such modifications are known and described, for example, in WO 2012/135805 and WO 2013/052523, the entire contents of which are incorporated herein by reference. Additional terminal modifications are described, for example, in WO 2014/164253 and WO 2016/011026, WO 2012/045075 and WO 2014/093924, the entire contents of which are incorporated herein by reference. Chimeric enzymes useful for synthesizing a capped RNA molecule that may include at least one chemical modification (e.g., modified mRNA) are described in WO 2014/028429, the entire contents of which are incorporated herein by reference.

In some cases, the modified mRNA may be cyclized or interlinked (concatemerized) to produce a translationally active molecule to aid in the interaction between the poly-A binding protein and the 5' binding protein. The mechanism of cyclization or concatemerization can be carried out by at least 3 different pathways, 1) chemical catalysis, 2) enzymatic catalysis, and 3) ribozyme catalysis. The newly formed 5'-/3' -linkage may be intramolecular or intermolecular. Such modifications are described, for example, in WO 2013/151736.

Methods of preparing and purifying modified RNAs are known and disclosed in the art. For example, modified RNAs are prepared using only In Vitro Transcription (IVT) enzyme synthesis. Methods of preparing IVT polynucleotides are known in the art and are described in WO 2013/151666、WO 2013/151668、WO 2013/151663、WO 2013/151669、WO 2013/151670、WO 2013/151664、WO 2013/151665、WO 2013/151671、WO 2013/151672、WO 2013/151667 and WO 2013/151736, the entire contents of which are incorporated herein by reference. The purification method comprises purifying an RNA transcript comprising a polyA tail by contacting the sample with a surface to which a plurality of thymidine or derivatives thereof and/or a plurality of uracil or derivatives thereof (polyT/U) are attached under conditions such that the RNA transcript is bound to the surface, and eluting the purified RNA transcript from the surface (WO 2014/152031), using ion (e.g. anion) exchange chromatography (WO 2014/144767) allowing isolation of longer RNA of up to 10,000 nucleotides in length via a scalable method, and subjecting the modified mRNA sample to DNase treatment (WO 2014/152030).

Formulations of modified RNAs are known and are described, for example, in WO 2013/090648. For example, the formulation may be, but is not limited to, nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipids, lipid complexes, liposomes, polymers, carbohydrates (including monosaccharides), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin adhesives, fibrinogen, thrombin, rapidly eliminated lipid nanoparticles (reLNP), and combinations thereof.

Modified RNAs encoding polypeptides in human diseases, antibodies, viruses and various in vivo environmental fields are known and disclosed in, for example, international publication nos. WO 2013/151666, WO 2013/151668, WO 2013/151663, WO 2013/151669, WO 2013/151670, WO 2013/151664, WO 2013/151665, table 6 of WO 2013/151736, tables 6 and 7 of international publication nos. WO 2013/151672, tables 6, 178 and 179 of international publication nos. WO 2013/151671, tables 6, 185 and 186 of international publication nos. WO 2013/151667, the entire contents of which are incorporated herein by reference. Any of the foregoing may be synthesized as an IVT polynucleotide, a chimeric polynucleotide, or a circular polynucleotide, and each may include one or more modified nucleotides or terminal modifications.

Inhibitory RNA

In some cases, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) includes an inhibitory RNA molecule that functions, for example, through an RNA interference (RNAi) pathway. In some cases, the inhibitory RNA molecules reduce the level of gene expression in the target organism and/or reduce the level of protein in the target organism. In some cases, the inhibitory RNA molecule inhibits gene expression. For example, the inhibitory RNA molecule may include short interfering RNAs or precursors thereof, short hairpin RNAs and/or micrornas or precursors thereof that target genes in the target organism. Certain RNA molecules can inhibit gene expression through biological processes of RNA interference (RNAi). RNAi molecules include RNA or RNA-like structures, which typically comprise 15-50 base pairs (e.g., about 18-25 base pairs) and have nucleobase sequences that are identical (or complementary) or nearly identical (or substantially complementary) to the coding sequences in target genes expressed in cells. RNAi molecules include, but are not limited to, short interfering RNAs (siRNAs), double-stranded RNAs (dsRNAs), short hairpin RNAs (shRNAs), partial duplex (meroduplex), dicer substrates, and multivalent RNA interference (U.S. Pat. Nos. 8,084,599, 8,349,809, 8,513,207, and 9,200,276, the entire contents of which are incorporated herein by reference). Inhibitory RNA molecules can be chemically synthesized or transcribed in vitro.

Additional examples of inhibitory RNA molecules include those described in detail in International patent application publication No. WO 2021/04301 (the entire contents of which are incorporated herein by reference).

Cyclic polyribonucleotides

LNMP/circRNA therapeutic compositions include cyclic polyribonucleotide molecules, such as circRNA encoding polypeptides. The cyclic polyribonucleotides comprise elements as described below and expression sequences encoding polypeptides. In some embodiments, the cyclic polyribonucleotides include any feature, or any combination of features, disclosed in international patent publication No. WO2019/118919 (the entire contents of which are incorporated herein by reference).

In some embodiments, the cyclic polynucleic acid is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

In some embodiments, the cyclic polyribonucleotide is 500 nucleotides to 20,000 nucleotides, 1,000 to 20,000 nucleotides, 2,000 to 20,000 nucleotides, or 5,000 to 20,000 nucleotides. In some embodiments, the cyclic polyribonucleotide is 500 nucleotides to 10,000 nucleotides, 1,000 to 10,000 nucleotides, 2,000 to 10,000 nucleotides, or 5,000 to 10,000 nucleotides.

Internal ribosome entry site

In some embodiments, the circular polyribonucleotides described herein include one or more Internal Ribosome Entry Site (IRES) elements. In some embodiments, the IRES is operably linked to one or more expression sequences (e.g., each IRES is operably linked to one or more expression sequences, wherein each expression sequence optionally encodes a polypeptide). In embodiments, the IRES is located between the heterologous promoter and the 5' end of the coding sequence (e.g., the coding sequence encoding a polypeptide).

IRES elements suitable for inclusion in polyribonucleotides (e.g., polyribonucleotide cargo) include RNA sequences that are capable of engaging eukaryotic ribosomes. In some embodiments, the IRES element is at least about 5 nt, at least about 8 nt, at least about 9 nt, at least about 10 nt, at least about 15 nt, at least about 20 nt, at least about 25 nt, at least about 30 nt, at least about 40 nt, at least about 50 nt, at least about 100 nt, at least about 200 nt, at least about 250 nt, at least about 350 nt, or at least about 500 nt.

In some embodiments, the IRES element is from DNA from an organism including, but not limited to, viruses, mammals, and Drosophila (Drosophila). Such viral DNA may be from, but is not limited to, picornaviral complementary DNA (cDNA), including encephalomyocarditis virus (EMCV) cDNA and poliovirus cDNA. In one embodiment, drosophila DNA from which IRES elements are derived includes, but is not limited to, the antennapedia mutant gene from Drosophila melanogaster.

In some embodiments, the IRES sequence is an IRES sequence selected from the group consisting of Taylor syndrome virus (Taura syndrome virus), trypsin virus (Triatoma virus), taylor encephalomyelitis virus (Theiler's encephalomyelitis virus), simian Virus 40 (simian Virus), formica Fusca Virus 1 (Solenopsis invicta virus 1), grandis tube aphid Virus (Rhopalosiphum padi virus), taylor's disease Virus (Theiler's encephalomyelitis virus), and combinations thereof, Reticuloendotheliosis virus (Reticuloendotheliosis virus), human poliovirus 1 (fuman poliovirus 1), proteus Li Yachun enterovirus (Plautia STALL INTESTINE virus), keshmifer bee virus (Kashmir bee virus), human rhinovirus 2 (Human rhinovirus, HRV-2), equipped with glass Virus 1 (Homalodisca coagulata virus-1), Human immunodeficiency virus type 1 (Human Immunodeficiency Virus type 1), eyew's disease virus 1 (Homalodisca coagulata virus-1), eyew's disease virus (Himetobi P virus), hepatitis C virus (HEPATITIS C virus), hepatitis A virus (HEPATITIS A virus), GB hepatitis virus (HEPATITIS GB VIRUS), foot and mouth disease virus (foot and mouth disease virus), and, Human enterovirus 71 (Human enterovirus) and equine rhinitis virus (Equine rhinitis virus), tea inchworm type picornavirus (Ectropis obliqua picorna-like virus), encephalomyocarditis virus (Encephalomyocarditis virus, EMCV), drosophila C virus (Drosophila C Virus), cruciferae tobacco mosaic virus (Crucifer tobamo virus), Cricket paralysis virus (CRICKET PARALYSIS virus), bovine viral diarrhea virus 1 (Bovine VIRAL DIARRHEA virus 1), black queen bee virus (Black Queen Cell Virus), aphid lethal paralysis virus (APHID LETHAL PARALYSIS virus), avian encephalomyelitis virus (Avian encephalomyelitis virus, AEV), acute bee paralysis virus (Acute bee paralysis virus), and, Hibiscus syriacus chlorotic ringspot virus (Hibiscus chlorotic ringspot virus), classical swine fever virus (CLASSICAL SWINE FEVER virus), human FGF2, human SFTPA1, human AML1/RUNX1, drosophila antennapedia mutation (Drosophila antennapedia), human AQP4, human AT1R, human BAG-l, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, Mouse NDST L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-L, mouse Rbm3, drosophilA reaper, canine (Canine) Scamper, drosophilA Ubx, human UNR, mouse UtrA, human VEGF-A, human XIAP, arenavirus (Salivirus), coxsackievirus (Cosavirus), paraenterovirus (Parechovirus), drosophilA hairless (DrosophilA hairless), Saccharomyces cerevisiae TFIID, saccharomyces cerevisiae YAP1, human c-src, human FGF-l, simian parvovirus (Simian picomavirus), turnip shrunken virus (Turnip crinkle virus), azfeldt-Jakob virus (Aichivirus), crohn virus (Crohivirus), echovirus 11, an aptamer to eIF4G, coxsackie virus B3 (CVB 3) or Coxsackie virus A (CVB 1/2). In yet another embodiment, the IRES is an IRES sequence of coxsackievirus B3 (CVB 3). In a further embodiment, the IRES is an IRES sequence of an encephalomyocarditis virus. In further embodiments, the IRES is an IRES sequence of a taylor encephalomyelitis virus.

The IRES sequence may have a modified sequence compared to the wild-type IRES sequence. In some embodiments, when the last nucleotide of the wild-type IRES is not a cytosine nucleic acid residue, the last nucleotide of the wild-type IRES sequence may be modified such that it is a cytosine residue. For example, the IRES sequence may be a CVB3 IRES sequence in which terminal adenosine residues are modified to cytosine residues. In some embodiments, the modified CVB3 IRES may have the following nucleic acid sequences:

。

In some embodiments, the IRES sequence is an enterovirus 71 (EV 17) IRES. In some embodiments, the terminal guanosine residue of the EV17 IRES sequence is modified to a cytosine residue. In some embodiments, the modified EV71 IRES can have the following nucleic acid sequence:

。

In some embodiments, a polyribonucleotide (e.g., polyribonucleotide cargo) includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5, or more) expression sequence. In some embodiments, the IRES flanks the at least one (e.g., 2, 3, 4, 5 or more) expression sequence. In some embodiments, a polyribonucleotide (e.g., polyribonucleotide cargo) includes one or more IRES sequences on one or both sides of each expressed sequence, resulting in separation of the resulting peptide and/or polypeptide. For example, a polyribonucleotide (e.g., polyribonucleotide cargo) described herein can include a first IRES operably linked to a first expression sequence (e.g., encoding a first polypeptide) and a second IRES operably linked to a second expression sequence (e.g., encoding a second polypeptide).

In some embodiments, a polyribonucleotide described herein (e.g., polyribonucleotide cargo) comprises an IRES (e.g., an IRES operably linked to a coding region). For example, polyribonucleotides (e.g., polyribonucleotide cargo) can include any IRES：Chen et al.Mol. Cell81(20):4300-4318, 2021;Jopling et al.Oncogene20:2664-2670, 2001;Baranick et al.PNAS105(12):4733-4738, 2008;Lang et al.Molecular Biology of the Cell13(5):1792-1801, 2002;Dorokhov et al.PNAS99(8):5301-5306, 2002;Wang et al.Nucleic Acids Research33(7):2248-2258, 2005;Petz et al.Nucleic Acids Research35(8):2473-2482, 2007, Chen et al. Science 268:415-417, 1995;Fan et al. Nature Communication 13(1):3751-3765, 2022 as described below and international publication No. WO2021/263124, the entire contents of each of which are incorporated herein by reference.

Regulatory element

In some embodiments, the cyclic polyribonucleotide includes a regulatory element, such as a sequence that alters expression of an expressed sequence within the cyclic polyribonucleotide. In some embodiments, a polyribonucleotide described herein (e.g., polyribonucleotide cargo) comprises one or more regulatory elements.

Regulatory elements may include sequences that are positioned adjacent to an expression sequence encoding an expression product (e.g., a polypeptide). The regulatory element may be operably linked to the adjacent sequence. The regulatory element may increase the amount of the expressed product as compared to the amount of the expressed product when the regulatory element is not present. Regulatory elements may be used to increase the expression of one or more polypeptides encoded by a polyribonucleotide (e.g., polyribonucleotide cargo). Likewise, regulatory elements may be used to reduce the expression of one or more polypeptides encoded by polyribonucleotides (e.g., polyribonucleotide cargo). In some embodiments, one regulatory element may be used to increase expression of one polypeptide and another regulatory element may be used to decrease expression of another polypeptide on the same polyribonucleotide (e.g., polyribonucleotide cargo). Furthermore, a regulatory element may increase the amount of a product (e.g., a polypeptide) expressed by a plurality of expression sequences connected in series. Thus, a regulatory element may enhance expression of one or more expression sequences. Multiple regulatory elements may also be used, for example to differentially regulate expression of different expression sequences.

In some embodiments, the regulatory element is a translational regulator. The translational regulator may regulate translation of the expressed sequence of the cyclic polyribonucleotide. The translational regulator may be a translational enhancer or a translational repressor. In some embodiments, the cyclic-polyribonucleotide includes at least one translational regulator adjacent to at least one expressed sequence. In some embodiments, the cyclic polyribonucleotides include a translational regulator adjacent to each expressed sequence. In some embodiments, a translational regulator is present on one or both sides of each expressed sequence, resulting in a separation of the expression products, e.g., polypeptides.

In some embodiments, the regulatory element is microRNA (miRNA) or a miRNA binding site.

Further examples of regulatory elements are described, for example, in paragraphs [0154] to [0161] of International patent publication No. WO2019/118919, the entire contents of which are incorporated herein by reference.

Signal sequence

In some embodiments, the polypeptide expressed by the cyclic or linear polyribonucleotides disclosed herein comprises a secreted protein, such as a protein that naturally comprises a signal sequence, or a protein that does not normally encode a signal sequence but is modified to comprise a signal sequence. In some embodiments, the polypeptide encoded by a circular or linear polyribonucleotide comprises a secretion signal. For example, the secretion signal may be a naturally encoded secretion signal of a secreted protein. In another example, the secretion signal may be a modified secretion signal of a secreted protein. In other embodiments, the polypeptide encoded by the circular or linear polyribonucleotide does not comprise a secretion signal.

In some embodiments, a polyribonucleotide (e.g., a cyclic polyribonucleotide, a linear polyribonucleotide, a polyribonucleotide cargo of a polyribonucleotide) encodes multiple copies (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) of the same polypeptide. In some embodiments, at least one copy of the polypeptide comprises a signal sequence and at least one copy of the polypeptide does not comprise a signal sequence. In some embodiments, a polyribonucleotide (e.g., a cyclic polyribonucleotide, a linear polyribonucleotide, a polyribonucleotide cargo of a polyribonucleotide) encodes a plurality of polypeptides (e.g., a plurality of different polypeptides or a plurality of polypeptides having less than 100% sequence identity), wherein at least one of the plurality of polypeptides comprises a signal sequence and at least one copy of the plurality of polypeptides does not comprise a signal sequence.

In some embodiments, the signal sequence is a wild-type signal sequence present at the N-terminus of the corresponding wild-type polypeptide, e.g., when expressed endogenously. In some embodiments, the signal sequence is heterologous to the polypeptide, e.g., the signal sequence is absent when the wild-type polypeptide is expressed endogenously. The polynucleic nucleotide sequence encoding the polypeptide may be modified to remove the nucleotide sequence encoding the wild-type signal sequence and/or to add a sequence encoding a heterologous signal sequence.

The polypeptide encoded by a polyribonucleotide may comprise a signal sequence that directs the polypeptide into the secretory pathway. In some embodiments, the signal sequence may direct the polypeptide to reside in certain organelles (e.g., endoplasmic reticulum, golgi apparatus, or endosomes). In some embodiments, the signal sequence directs secretion of the polypeptide from the cell. For secreted proteins, the signal sequence may be cleaved after secretion to yield the mature protein. In other embodiments, the signal sequence may be embedded in the membrane of a cell or some organelle, creating a transmembrane segment that anchors the protein to the membrane of the cell, endoplasmic reticulum, or golgi apparatus. In certain embodiments, the signal sequence of the transmembrane protein is a short sequence at the N-terminus of the polypeptide. In other embodiments, the first transmembrane domain serves as a first signal sequence that targets the protein to the membrane. In some embodiments, the polypeptide encoded by a polyribonucleotide comprises a secretion signal sequence or a transmembrane insertion signal sequence, or does not comprise a signal sequence.

Cleavage domain

The cyclic polyribonucleotides of the present disclosure may comprise a cleavage domain (e.g., a staggered (stagger) element or cleavage sequence).

The term "staggered element" refers to a portion, e.g., a nucleotide sequence, that induces a ribosome pause during translation. In some embodiments, the staggered elements are non-conserved amino acid sequences with strong alpha helix propensity, followed by consensus sequence-D (V/I) ExNPGP, where x = any amino acid (SEQ ID NO: 16). In some embodiments, the staggered elements may comprise chemical moieties, such as glycerol, non-nucleic acid linking moieties, chemical modifications, modified nucleic acids, or any combination thereof.

In some embodiments, the cyclic polyribonucleotide comprises at least one staggered element adjacent to the expressed sequence. In some embodiments, the cyclic polyribonucleotides comprise a staggered element adjacent to each expressed sequence. In some embodiments, staggered elements are present on one or both sides of each expressed sequence, resulting in separation of the expression products, e.g., peptides and or polypeptides. In some embodiments, the staggered elements are part of one or more expressed sequences. In some embodiments, the circular polyribonucleotide comprises one or more expression sequences, and each of the one or more expression sequences is separated from the subsequent expression sequences by a staggered element on the circular polyribonucleotide. In some embodiments, the staggering element prevents (a) the production of a single polypeptide from two rounds of translation of a single expressed sequence or (b) the production of a single polypeptide from one or more rounds of translation of two or more expressed sequences. In some embodiments, the staggered elements are sequences independent of one or more expressed sequences. In some embodiments, the interlaced elements comprise a portion of one of the one or more expression sequences.

In some embodiments, the circular polyribonucleotides comprise staggered elements. To avoid the production of continuous expression products such as peptides or polypeptides, while maintaining rolling circle translation, staggered elements may be included to induce ribosome stalls during translation. In some embodiments, the staggered element is located 3' of at least one of the one or more expression sequences. The interleaving element may be configured to arrest ribosomes during rolling circle translation of the cyclic polyribonucleotide. The staggered elements may include, but are not limited to, a 2A-like or CHYSEL (SEQ ID NO: 24) (cis-acting hydrolase element) sequence. In some embodiments, the staggered elements encode a sequence having the C-terminal consensus sequence X1X2X3EX5NPGP (SEQ ID NO: 38), wherein X1 is absent or G or H, X2 is absent or D or G, X3 is D or V or I or S or M, and X5 is any amino acid. In some embodiments, the sequence comprises a non-conserved amino acid sequence with a strong alpha helix propensity, followed by consensus sequence-D (V/I) EXNPGP (SEQ ID NO: 39), wherein x = any amino acid. Some non-limiting examples of interlaced elements include

GDIEQNPGP (SEQ ID NO: 57) and DSEFNPGP (SEQ ID NO: 58).

In some embodiments, the staggered elements described herein cleave the expression product, e.g., between G and P of the consensus sequences described herein. As one non-limiting example, a circular polyribonucleotide comprises at least one staggered element to cleave the expression product. In some embodiments, the cyclic polyribonucleotide comprises a staggered element adjacent to at least one expressed sequence. In some embodiments, the circular polyribonucleotides comprise staggered elements after each expressed sequence. In some embodiments, the circular polyribonucleotides comprise staggered elements present on one or both sides of each expressed sequence, resulting in translation of a single peptide and or polypeptide per expressed sequence.

In some embodiments, the staggering element comprises one or more modified nucleotides or unnatural nucleotides that induce a ribosome pause during translation. Non-natural nucleotides may include Peptide Nucleic Acids (PNAs), morpholino and Locked Nucleic Acids (LNAs), as well as ethylene Glycol Nucleic Acids (GNAs) and Threose Nucleic Acids (TNAs). Examples are those that distinguish from naturally occurring DNA or RNA by altering the molecular backbone, for example. Exemplary modifications may include any modification to a sugar, nucleobase, internucleoside linkage (e.g., linking a phosphate/phosphodiester linkage/phosphodiester backbone), and any combination that may induce ribosome pauses during translation. Some exemplary modifications provided herein are described elsewhere herein.

In some embodiments, the staggered elements are present in other forms in the circular polyribonucleotide. For example, in some exemplary cyclic polyribonucleotides, the staggered element comprises a termination element of the first expression sequence in the cyclic polyribonucleotide, and a nucleotide spacer sequence separating the termination element from the first translation initiation sequence of expression following the first expression sequence. In some examples, the first staggered element of the first expression sequence is located upstream (5') of the first translation initiation sequence of expression following the first expression sequence in the cyclic polyribonucleotide. In some cases, the first expression sequence and the expression sequence following the first expression sequence are two independent expression sequences in a cyclic polyribonucleotide. The distance between the first interleaving element and the first translation initiation sequence may enable a continuous translation of the first expression sequence and the following expression sequence.

In some embodiments, the first interleaving element comprises a termination element and separates the expression product of the first expression sequence from the expression product of the expression sequence following it, thereby producing discrete expression products. In some cases, a circular polyribonucleotide comprising a first interleaving element upstream of a first translation initiation sequence of a subsequent sequence in the circular polyribonucleotide is translated consecutively, while a corresponding circular polyribonucleotide comprising an interleaving element of a second expression sequence upstream of a second translation initiation sequence of an expression sequence subsequent to the second expression sequence is not translated consecutively. In some cases, there is only one expression sequence in the circular polyribonucleotide, and the first expression sequence and subsequent expression sequences are the same expression sequences. In some exemplary cyclic polyribonucleotides, the staggered element includes a first termination element of the first expression sequence in the cyclic polyribonucleotide, and a nucleotide spacer sequence that separates the termination element from downstream translation initiation sequences. In some such examples, the first interleaving element is located upstream (5') of the first translation initiation sequence of the first expression sequence in the cyclic polyribonucleotide. In some cases, the distance between the first interleaving element and the first translation initiation sequence may enable continuous translation of the first expression sequence and subsequently the expression sequence.

In some embodiments, the first interleaving element separates one round of expression products of the first expression sequence from the next round of expression products of the first expression sequence, thereby producing discrete expression products. In some cases, a circular polyribonucleotide comprising a first interleaving element upstream of a first translation initiation sequence of a first expression sequence in a circular polyribonucleotide is translated consecutively, while a corresponding circular polyribonucleotide comprising an interleaving element upstream of a second translation initiation sequence of a second expression sequence in a corresponding circular polyribonucleotide is not translated consecutively. In some cases, the distance between the second staggered element in the corresponding circular polyribonucleotide and the second translation initiation sequence is at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x of the distance between the first staggered element in the circular polyribonucleotide and the first translation initiation point. In some cases, the distance between the first interlaced element and the first translation initiation point is at least 2 nt、3 nt、4 nt、5 nt、6 nt、7 nt、8 nt、9 nt、10 nt、11 nt、12 nt、13 nt、14 nt、15 nt、16 nt、17 nt、18 nt、19 nt、20 nt、25 nt、30 nt、35 nt、40 nt、45 nt、50 nt、55 nt、60 nt、65 nt、70 nt、75 nt or greater. In some embodiments, the distance between the second interlaced element and the second translation initiation point is at least 2 nt、3 nt、4 nt、5 nt、6 nt、7 nt、8 nt、9 nt、10 nt、11 nt、12 nt、13 nt、14 nt、15 nt、16 nt、17 nt、18 nt、19 nt、20 nt、25 nt、30 nt、35 nt、40 nt、45 nt、50 nt、55 nt、60 nt、65 nt、70 nt、75 nt or greater than the distance between the first interlaced element and the first translation initiation point. In some embodiments, the cyclic polyribonucleotide comprises more than one expression sequence.

Examples of interlaced elements are described in paragraphs [0172] - [0175] of International patent publication No. WO2019/118919 (the entire contents of which are incorporated herein by reference).

In some embodiments, multiple polypeptides encoded by a cyclic ribonucleotide may be separated by an IRES between each polypeptide (e.g., each polypeptide is operably linked to a separate IRES). For example, a cyclic polyribonucleotide may include a first IRES operably linked to a first expression sequence and a second IRES operably linked to a second expression sequence. The IRES between all polypeptides may be the same IRES. IRES may vary between different polypeptides.

In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide. For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, 2A, and a second polypeptide.

In some embodiments, the plurality of polypeptides may be separated by a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide.

In some embodiments, the plurality of polypeptides may be separated by a 2A self-cleaving peptide and a protease cleavage site (e.g., a furin cleavage site). For example, a circular polyribonucleotide may encode an IRES operably linked to an open reading frame encoding a first polypeptide, 2A, a protease cleavage site (e.g., a furin cleavage site), and a second polypeptide. The circular polyribonucleotide may also encode an IRES operably linked to open reading frames encoding the first polypeptide, a protease cleavage site (e.g., a furin cleavage site), 2A, and the second polypeptide. The tandem 2A and furin cleavage sites may be referred to as furin-2A (which includes furin-2A or 2A-furin, arranged in either orientation).

In addition, multiple polypeptides encoded by cyclic ribonucleotides may be separated by IRES and 2A sequences. For example, an IRES may be located between one polypeptide and a second polypeptide, and a 2A peptide may be located between the second polypeptide and a third polypeptide. The selection of a particular IRES or 2A self-cleaving peptide may be used to control the level of expression of a polypeptide under the control of an IRES or 2A sequence. For example, depending on the IRES and or 2A peptide selected, the expression of the polypeptide may be higher or lower.

In some embodiments, the cyclic polyribonucleotide comprises at least one cleavage sequence. In some embodiments, the cleavage sequence is adjacent to the expression sequence. In some embodiments, the cleavage sequence is located between two expression sequences. In some embodiments, the cleavage sequence is comprised in an expression sequence. In some embodiments, the cyclic polyribonucleotide comprises 2 to 10 cleavage sequences. In some embodiments, the cyclic polyribonucleotide comprises 2 to 5 cleavage sequences. In some embodiments, a plurality of cleavage sequences are located between a plurality of expression sequences, e.g., a circular polyribonucleotide can comprise three expression sequences and two cleavage sequences such that there is one cleavage sequence between each expression sequence. In some embodiments, the circular polyribonucleotide comprises a cleavage sequence, e.g., in self-destructing (immolating) or cleavable or self-cleaving circRNA. In some embodiments, the cyclic polyribonucleotide comprises two or more cleavage sequences, resulting in the separation of the cyclic polyribonucleotide into multiple products, e.g., miRNA, linear RNA, smaller cyclic polyribonucleotides, and the like.

In some embodiments, the cleavage sequence comprises a ribozyme RNA sequence. Ribozymes (derived from ribonucleases, also known as rnases or catalytic RNAs) are an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze the hydrolysis of one of their own phosphodiester bonds or catalyze the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of ribosomes. Catalytic RNAs can be "evolved" by in vitro methods. Similar to riboswitch activity discussed above, ribozymes and their reaction products can regulate gene expression. In some embodiments, the catalytic RNA or ribozyme may be placed within a larger non-coding RNA such that the ribozyme is present in many copies within the cell in order to chemically convert molecules in bulk. In some embodiments, both the aptamer and the ribozyme may be encoded in the same non-coding RNA.

In some embodiments, the cleavage sequence encodes a cleavable polypeptide linker. For example, a cyclic polyribonucleotide may encode two or more polypeptides, e.g., wherein the two or more polypeptides are encoded by a single Open Reading Frame (ORF). For example, two or more polypeptides may be encoded by a single open reading frame, the expression of which is controlled by an IRES. In some embodiments, the ORFs also encode polypeptide linkers, e.g., such that the expression products of the ORFs encode two or more polypeptides, each separated by a sequence encoding a polypeptide linker (e.g., a 5-200, 5-100, 5-50, 5-20, 50-100, or 50-200 amino acid linker). The polypeptide linker can comprise a cleavage site, e.g., a cleavage site that is recognized and cleaved by a protease (e.g., an endogenous protease in a subject after administration of a cyclic polyribonucleotide to the subject). In such embodiments, a single expression product comprising the amino acid sequences of two or more polypeptides is cleaved upon expression such that the two or more polypeptides are separated upon expression. Exemplary protease cleavage sites are known to those of skill in the art, for example, as the amino acid sequence of a protease cleavage site recognized by a metalloprotease (e.g., a Matrix Metalloprotease (MMP), such as any one or more of MMP 1-28), a depolymerizing agent, and a metalloprotease (ADAM, such as any one or more of ADAM 2, 7-12, 15, 17-23, 28-30, and 33), a serine protease (e.g., furin), a urokinase type plasminogen activator, a proteolytic enzyme, a cysteine protease, an aspartic protease, or a cathepsin. In some embodiments, the protease is MMP9 or MMP2. In some embodiments, the protease is a proteolytic enzyme.

In some embodiments, the cyclic polyribonucleotides described herein are self-destructing cyclic polyribonucleotides, cleavable cyclic polyribonucleotides, or self-cleaving cyclic polyribonucleotides. The circular polyribonucleotide can deliver cellular components including, for example, RNA, long non-coding RNA (lncRNA), long intergenic non-coding RNA (lincRNA), microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), non-coding RNA (ncRNA), small interfering RNA (siRNA), or small hairpin RNA (shRNA). In some embodiments, the circular polyribonucleotides comprise mirnas that are separated by (i) a self-cleaving element, (ii) a cleavage recruitment site, (iii) a degradable linker, (iv) a chemical linker, and/or (v) a spacer sequence. In some embodiments, the circRNA comprises siRNAs that are separated by (i) a self-cleaving element, (ii) a cleavage recruitment site (e.g., ADAR), (iii) a degradable linker (e.g., glycerol), (iv) a chemical linker, and/or (v) a spacer sequence. Non-limiting examples of self-cleaving elements include hammerhead ribozymes, splice element ribozymes, hairpin (hairpin) ribozymes, hepatitis Delta Virus (HDV) ribozymes, varkud Satellite (VS) ribozymes, and glmS ribozymes.

Translation initiation sequences

In some embodiments, a circular polyribonucleotide described herein comprises at least one translation initiation sequence. In some embodiments, a polyribonucleotide (e.g., polyribonucleotide cargo) comprises a translation initiation sequence operably linked to an expression sequence.

In some embodiments, the polyribonucleotide encodes a polypeptide and may comprise a translation initiation sequence, such as an initiation codon. In some embodiments, the translation initiation sequence comprises a Kozak or Shine-Dalgarno sequence. In some embodiments, the polyribonucleotide comprises a translation initiation sequence, such as a Kozak sequence, adjacent to the expression sequence. In some embodiments, the translation initiation sequence is a non-coding initiation codon. In some embodiments, a translation initiation sequence, such as a Kozak sequence, is present on one or both sides of each expression sequence, resulting in separation of the expression products. In some embodiments, the polyribonucleotide comprises at least one translation initiation sequence adjacent to the expression sequence. In some embodiments, the translation initiation sequence provides conformational flexibility to the polyribonucleotide. In some embodiments, the translation initiation sequence is located within a substantially single stranded region of a polyribonucleotide. Further examples of translation initiation sequences are described in paragraphs [0163] - [0165] of International patent publication No. WO2019/118919, the entire contents of which are incorporated herein by reference.

The polyribonucleotide may comprise more than 1 initiation codon, for example, but not limited to, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60, or more than 60 initiation codons. Translation may begin at the first start codon, or may begin downstream of the first start codon.

In some embodiments, the polyribonucleotide may begin at a codon that is not the first start codon, e.g., AUG. Translation of the polyribonucleotide may begin at an alternative translation initiation sequence (e.g., without limitation ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG). In some embodiments, translation begins at an alternative translation initiation sequence under selective conditions (e.g., stress-induced conditions). As one non-limiting example, translation of a polyribonucleotide may begin at an alternative translation initiation sequence, such as ACG. As another non-limiting example, polyribonucleotide translation may begin at the alternative translation initiation sequence CTG/CUG. As another non-limiting example, polyribonucleotide translation may begin at the alternative translation initiation sequence GTG/GUG. As another non-limiting example, a polyribonucleotide may begin translation at a repeat-related non-AUG (RAN) sequence, such as an alternative translation initiation sequence comprising a short segment of a repeat RNA (stretch) (e.g., CGG, GGGGCC (SEQ ID NO: 93), CAG, CTG).

Termination element

In some embodiments, a polyribonucleotide (e.g., polyribonucleotide cargo) described herein comprises at least one terminating element. In some embodiments, the polyribonucleotide comprises a termination element operably linked to the expression sequence. In some embodiments, the polynucleotide lacks a termination element.

In some embodiments, the polyribonucleotide comprises one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the polyribonucleotide comprises one or more expressed sequences, and the expressed sequences lack a termination element, such that the polyribonucleotide is translated serially. The absence of a termination element may result in rolling circle translation or continuous expression of the expression product.

In some embodiments, the circular polyribonucleotides comprise one or more expression sequences, and each expression sequence may or may not have a termination element. In some embodiments, the cyclic polyribonucleotide comprises one or more expression sequences, and the expression sequences lack a termination element, such that the cyclic polyribonucleotide is continuously translated. The absence of a termination element may result in rolling circle translation or continuous expression of an expression product, such as a peptide or polypeptide, due to lack of ribosome arrest or shedding. In such embodiments, rolling circle translation expresses a contiguous expression product through each expression sequence. In some other embodiments, the termination element of the expression sequence may be part of an interleaving element. In some embodiments, one or more expression sequences in a cyclic polyribonucleotide comprise a termination element. However, subsequent (e.g., second, third, fourth, fifth, etc.) rolling circle translation or expression of the expressed sequence is performed in the circular polyribonucleotide. In such cases, when the ribosome encounters a termination element, such as a termination codon, the expression product may be detached from the ribosome and translation terminated. In some embodiments, translation is terminated when the ribosome (e.g., at least one subunit of the ribosome) remains in contact with the cyclic polyribonucleotide.

In some embodiments, the cyclic polyribonucleotides comprise a termination element at the end of one or more expression sequences. In some embodiments, one or more expression sequences comprise two or more termination elements in succession. In such embodiments, translation is terminated and rolling circle translation is terminated. In some embodiments, the ribosome is completely detached from the cyclic polyribonucleotide. In some such embodiments, the generation of subsequent (e.g., second, third, fourth, fifth, etc.) expression sequences in the cyclic polyribonucleotide may require that the ribosome be re-conjugated to the cyclic polyribonucleotide before translation begins. Typically, the termination element comprises an in-frame nucleotide triplet, e.g., UAA, UGA, UAG, that signals translation termination. In some embodiments, one or more of the termination elements in the circular polyribonucleotide are frame shift termination elements, such as, but not limited to, out of frame or-1 and +1 frame shift reading frames (e.g., hidden terminators) that can terminate translation. The frameshift termination element includes nucleotide triplets TAA, TAG and TGA that appear in the second and third reading frames of the expressed sequence. The frameshift termination element may be important to prevent misreading of mRNA, which is often detrimental to cells. In some embodiments, the termination element is a stop codon.

Further examples of termination elements are described in paragraphs [0169] - [0170] of International patent publication No. WO2019/118919, the entire contents of which are incorporated herein by reference.

Spacer sequence

In some embodiments, a polyribonucleotide (e.g., a cyclic polyribonucleotide, a linear polyribonucleotide) described herein comprises one or more spacer sequences. A spacer or spacer sequence refers to any contiguous nucleotide sequence (e.g., any contiguous nucleotide sequence of one or more nucleotides) that provides distance or flexibility between two adjacent polynucleotide regions. The spacer may be present between any of the nucleic acid elements described herein. The spacer may also be present within a nucleic acid element as described herein.

For example, wherein the nucleic acid comprises any two or more of (A) a 3 'catalytic intron fragment, (B) a 3' splice site, (C) a 3 'exon fragment, (D) a polynucleic nucleotide cargo, (E) a 5' exon fragment, (F) a 5 'splice site, and (G) a 5' catalytic intron fragment, a spacer region may be present between any one or more of the elements. As described herein, any of elements (a), (B), (C), (D), (E), (F), or (G) may be separated by a spacer sequence. For example, there may be a spacer between (A) and (B), between (B) and (C), between (C) and (D), between (D) and (E), between (E) and (F), or between (F) and (G).

In some embodiments, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) further comprises a first spacer sequence between the 5' exon fragment of (C) and the polyribonucleotide cargo of (D). The spacer may be, for example, at least 5 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, the polyribonucleotide further comprises a second spacer sequence between the polyribonucleotide cargo of (D) and the 5' exon fragment of (E).

Spacer sequences may be used to separate the IRES from adjacent structural elements to preserve the structure and function of the IRES or adjacent elements. The spacer may be specifically designed according to IRES. In some embodiments, RNA folding computer software such as RNAFold may be used to direct the design of the various elements (including spacers) of the vector. Thus, in one embodiment, the spacer sequence is located between the IRES and the 3 'or 5' exon fragment. In other embodiments, the spacer sequence is located between the expression sequence and the 3' exon fragment. In other embodiments, the spacer sequence is adjacent to the 5 'exon fragment or the 3' exon fragment.

The spacer may be, for example, at least 3 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. In some embodiments, each spacer sequence is at least 3 (e.g., at least 10, at least 15, at least 20) ribonucleotides in length. The length of each spacer region may be, for example, 5 to 800 (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800) ribonucleotides. In some embodiments, the spacer sequence is at least about 60 ribonucleotides in length. In some embodiments, the spacer sequence is about 60 to about 650 ribonucleotides in length.

In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence may comprise a poly (X) sequence. In some embodiments, the first spacer sequence and the second spacer sequence, or the first spacer sequence and the second spacer sequence, may comprise a poly (a) sequence. The first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence may comprise a poly (a-C) sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence comprise a poly (a-G) sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence comprise a poly (a-T) sequence. In some embodiments, the first spacer sequence, the second spacer sequence, or the first spacer sequence and the second spacer sequence comprise random sequences.

Spacers may also be present within the nucleic acid regions described herein. For example, a polynucleotide cargo region may comprise one or more spacers. The spacer may separate regions within the polynucleotide cargo.

In some embodiments, the spacer sequence may be, for example, at least 10 nucleotides, at least 15 nucleotides, or at least 70 nucleotides in length. In some embodiments, the spacer sequence is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length. In some embodiments, the spacer sequence is no more than 800, 700, 600, 500, 400, 300, 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides in length. In some embodiments, the spacer sequence is 20 to 70 nucleotides in length. In certain embodiments, the spacer sequence is 10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69 or 70 nucleotides in length.

The spacer sequence may be a poly (X) sequence, a poly (A-T) sequence, a poly (A-U) sequence, a poly (A-C) sequence, a poly (A-G) sequence, a poly (G), a poly (C) sequence, a poly (U) sequence, or a random sequence.

Exemplary spacer sequences are described in paragraphs [0293] to [0302] of International patent publication No. WO2019/118919 (the entire contents of which are incorporated herein by reference).

In some embodiments, the cyclic polyribonucleotide comprises a5 'spacer sequence (e.g., between the 5' annealing region and the polyribonucleotide cargo). In some embodiments, the 5' spacer sequence is at least about 60 nucleotides in length. In another embodiment, the 5' spacer sequence is at least 100 nucleotides in length. In a further embodiment, the 5' spacer sequence is at least about 200 nucleotides in length. In other embodiments, the 5' spacer sequence is at least 300 nucleotides in length. In another embodiment, the 5' spacer sequence is at least about 400 nucleotides in length. In another embodiment, the 5' spacer sequence is at least about 500 nucleotides in length. In other embodiments, the 5' spacer sequence is at least about 600 nucleotides in length. In other embodiments, the 5' spacer sequence is at least 700 nucleotides in length. In one embodiment, the 5' spacer is a poly (X) sequence. In one embodiment, the 5' spacer sequence is a poly (A) sequence. In another embodiment, the 5' spacer sequence is a poly (A-C) sequence. In some embodiments, the 5' spacer sequence comprises a poly (A-G) sequence. In some embodiments, the 5' spacer sequence comprises a poly (A-U) sequence. In some embodiments, the 5' spacer sequence comprises a random sequence.

In some embodiments, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) comprises a 3 'spacer sequence (e.g., between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the polyribonucleotide comprises a 3 'spacer sequence (e.g., between the 3' annealing region and the polyribonucleotide cargo). In some embodiments, the 3' spacer sequence is at least about 60 nucleotides in length. In another embodiment, the 3' spacer sequence is at least 100 nucleotides in length. In a further embodiment, the 3' spacer sequence is at least about 200 nucleotides in length. In other embodiments, the 3' spacer sequence is at least 300 nucleotides in length. In another embodiment, the 3' spacer sequence is at least about 400 nucleotides in length. In another embodiment, the 3' spacer sequence is at least about 500 nucleotides in length. In other embodiments, the 3' spacer sequence is at least about 600 nucleotides in length. In other embodiments, the 3' spacer sequence is at least 700 nucleotides in length. In one embodiment, the 3' spacer sequence is a poly (X) sequence. In one embodiment, the 3' spacer sequence is a poly (A) sequence. In another embodiment, the 3' spacer sequence is a poly (A-C) sequence. In some embodiments, the 3' spacer sequence comprises a poly (A-G) sequence. In some embodiments, the 3' spacer sequence comprises a poly (A-U) sequence. In some embodiments, the 3' spacer sequence comprises a random sequence.

In one embodiment, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) comprises a 5 'spacer sequence, but does not comprise a 3' spacer sequence. In another embodiment, the cyclic polyribonucleotide comprises a 3 'spacer sequence, but does not comprise a 5' spacer sequence. In another embodiment, the polyribonucleotide comprises neither a 5 'spacer sequence nor a 3' spacer sequence. In another embodiment, the polyribonucleotide does not comprise an IRES sequence. In further embodiments, the polyribonucleotide does not comprise an IRES sequence, a 5 'spacer sequence, or a 3' spacer sequence.

In some embodiments, the spacer sequence comprises at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, at least about 1000 ribonucleotides, or at least about ribonucleotides.

In some embodiments, the circular polyribonucleotide comprises a spacer sequence. In some embodiments, the linear polyribonucleotide comprises a spacer sequence. In some embodiments, the spacer sequence may be included upstream of the translation initiation sequence of the expression sequence. In some embodiments, the spacer sequence may be included downstream of the expression sequence. In some cases, one spacer of a first expression sequence is identical to or contiguous with or overlaps with another spacer of a second expression sequence. In some embodiments, the intron is a human intron. In some embodiments, the intron is a full length human intron, e.g., ZKSCAN1.

Exemplary spacer sequences are described in paragraphs [0197] - [201] of International patent publication No. WO2019/118919 (the entire contents of which are incorporated herein by reference).

In some embodiments, the cyclic polyribonucleotide comprises a poly (a) sequence. In some embodiments, the linear polyribonucleotide comprises a poly (a) sequence. Exemplary poly (A) sequences are described in paragraphs [0202] - [0205] of International patent publication No. WO2019/118919, the entire contents of which are incorporated herein by reference. In some embodiments, the cyclic polyribonucleotide lacks a poly (a) sequence. In some embodiments, the linear polyribonucleotide lacks a poly (a) sequence.

In some embodiments, the polyribonucleotide (e.g., cyclic polyribonucleotide, linear polyribonucleotide) comprises a spacer sequence into which one or more adenosine and uridine fragments are embedded. These AU-rich features can increase turnover of the expression product.

Introduction, removal or modification of spacer sequence enriched AU elements (ARE) can be used to modulate the stability or immunogenicity (e.g., the level of one or more markers of an immune or inflammatory response) of a cyclic polyribonucleotide. When designing a particular polyribonucleotide, one or more copies of an ARE can be introduced into the cyclic polyribonucleotide, and the copies of an ARE can regulate translation and/or production of the expression product. Similarly, ARE can be identified and removed or engineered into a cyclic polyribonucleotide to modulate intracellular stability, thereby affecting translation and production of the resulting protein.

It will be appreciated that any spacer from any gene may be incorporated into the corresponding flanking region of the cyclic polyribonucleotide.

In some embodiments, the cyclic polyribonucleotide lacks a 5' spacer sequence and is capable of protein expression from one or more of its expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a 3' spacer sequence and is capable of protein expression from one or more of its expression sequences. In some embodiments, the circular polyribonucleotide lacks a poly (a) sequence and is capable of protein expression from one or more of its expression sequences. In some embodiments, the circular polyribonucleotide lacks a termination element and is capable of protein expression from one or more of its expression sequences. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site and is capable of protein expression from one or more of its expression sequences. In some embodiments, the cyclic polyribonucleotide lacks a cap and is capable of protein expression from one or more of its expression sequences. In some embodiments, the circular polyribonucleotide lacks a 5 'spacer sequence, a 3' spacer sequence, and an IRES, and is capable of protein expression from one or more of its expression sequences. In some embodiments, the circular polyribonucleotides comprise one or more of a sequence encoding one or more miRNAs, a sequence encoding one or more replication proteins, a sequence encoding a foreign gene, a sequence encoding a therapeutic agent, a regulatory element (e.g., a translational regulator (e.g., a translational enhancer or repressor)), a translation initiation sequence, one or more regulatory nucleic acids (e.g., siRNA, lncRNA, shRNA) that target an endogenous gene, and a sequence encoding a therapeutic mRNA or protein.

In some embodiments, the cyclic polyribonucleotide lacks a 5' spacer sequence. In some embodiments, the circular polyribonucleotide lacks a spacer sequence. In some embodiments, the cyclic polyribonucleotide lacks a poly (X) sequence. In some embodiments, the circular polyribonucleotide lacks a termination element. In some embodiments, the circular polyribonucleotide lacks an internal ribosome entry site. In some embodiments, the cyclic polyribonucleotide lacks sensitivity to degradation by exonuclease. In some embodiments, the fact that the cyclic polyribonucleotide lacks susceptibility to degradation may indicate that the cyclic polyribonucleotide is not degraded by an exonuclease or only to a limited extent in the presence of an exonuclease, e.g., comparable or similar to in the absence of an exonuclease. In some embodiments, the cyclic polyribonucleotide is not degraded by exonucleases. In some embodiments, the cyclic polyribonucleotide has reduced degradation upon exposure to an exonuclease. In some embodiments, the cyclic polyribonucleotide lacks binding to a cap binding protein. In some embodiments, the cyclic polyribonucleotide lacks a 5' cap.

Generation of cyclic polyribonucleotides

The cyclic polyribonucleotides can be prepared according to any available technique, including but not limited to recombinant techniques and chemical synthesis. For example, a DNA molecule for producing a circular RNA molecule can include a DNA sequence of a naturally occurring original nucleic acid sequence, a modified version thereof, or a DNA sequence encoding a synthetic polypeptide (e.g., a chimeric molecule or fusion protein) that is not normally found in nature. The DNA and RNA molecules can be modified using a variety of techniques including, but not limited to, classical mutagenesis techniques and recombinant techniques, such as site-directed mutagenesis, chemical treatment of nucleic acid molecules to induce mutations, restriction endonuclease cleavage of nucleic acid fragments, ligation of nucleic acid fragments, polymerase Chain Reaction (PCR) amplification or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof.

In some embodiments, the linear polyribonucleotides used for circularization may be circularized or concatemerized. In some embodiments, the linear polyribonucleotides for cyclization can be cyclized in vitro prior to formulation and/or delivery. In some embodiments, the cyclic polyribonucleotide can be mixed with linear polyribonucleotides. In some embodiments, the linear polyribonucleotide has the same nucleic acid sequence as the cyclic polyribonucleotide.

In some embodiments, the linear polyribonucleotides used for cyclization are cyclized or concatenated using chemical methods to form a cyclic polyribonucleotide. In some chemical methods, the 5 'and 3' ends of a nucleic acid (e.g., linear polyribonucleotides for cyclization) contain chemically reactive groups that, when brought into proximity, can form new covalent bonds between the 5 'and 3' ends of the molecule. The 5 'end may comprise a NHS ester reactive group and the 3' end may comprise a3 '-amino-terminated nucleotide such that in an organic solvent, the 3' -amino-terminated nucleotide at the 3 'end of the linear RNA molecule will nucleophilic attack the 5' -NHS ester moiety to form a new 5'-/3' -amide bond. Other chemical methods of cyclization include, but are not limited to, click chemistry (e.g., alkyne and azide based methods, or clickable bases), olefin metathesis, phosphoramide linkage, hemi (hemiaminal) -imine cross-linking, base modification, and any combination thereof.

In some embodiments, the linear primary construct or linear polyribonucleotide can be circularized or concatemerized by methods such as chemical, enzymatic, splint ligation, or ribozyme-catalyzed methods to produce a circular polyribonucleotide. The newly formed 5'-3' bond may be an intramolecular bond or an intermolecular bond. For example, a splint ligase such as SplintR. Sup. Ligase, RNA ligase II, T4 RNA ligase or T4 DNA ligase may be used for splint ligation. According to this method, a single-stranded polynucleotide (splint), such as single-stranded DNA or RNA, can be designed to hybridize to both ends of a linear polyribonucleotide such that the two ends can be juxtaposed upon hybridization to the single-stranded splint. Thus, the splint ligase may catalyze the ligation of the two juxtaposed ends of a linear polyribonucleotide to produce a circular RNA. In some embodiments, DNA or RNA ligase may be used to synthesize the circular polynucleotide. As a non-limiting example, the ligase may be a circularized ligase or a circular ligase.

In another example, the 5 'or 3' end of the linear polyribonucleotide can encode a ligase ribozyme sequence such that during in vitro transcription, the resulting linear circRNA comprises an active ribozyme sequence that is capable of ligating the 5 'end of the linear polyribonucleotide with the 3' end of the linear polyribonucleotide. The ligase ribozyme may be derived from a type I intron, hepatitis delta virus, hairpin ribozyme, or may be selected by SELEX (ligand index enrichment system evolution).

In another example, the linear polyribonucleotides may be circularized or concatemerized by the use of at least one non-nucleic acid moiety. For example, at least one non-nucleic acid moiety may react with a region or feature near the 5 'end or near the 3' end of a linear polyribonucleotide to circularize or concatemerize the linear polyribonucleotide. In another example, the at least one non-nucleic acid moiety may be located at or linked to the 5 'or 3' end of the linear polyribonucleotide or near the 5 'or 3' end of the linear polyribonucleotide. The non-nucleic acid portion may be homologous or heterologous. As one non-limiting example, the non-nucleic acid moiety may be a bond, such as a hydrophobic bond, an ionic bond, a biodegradable bond, or a cleavable bond. As another non-limiting example, the non-nucleic acid moiety is a linking moiety. As yet another non-limiting example, the non-nucleic acid moiety may be an oligonucleotide or peptide moiety, such as an aptamer or non-nucleic acid linker as described herein.

In another example, linear polyribonucleotides can be cyclized or interlinked by self-splicing. In some embodiments, the linear polyribonucleotide may comprise an E-ring sequence for self-ligation. In another embodiment, the linear polyribonucleotide may comprise self-circularising introns such as 5 'and 3' splice junctions, or self-circularising catalytic introns such as type I, type II or type III introns. Non-limiting examples of type I intron self-splicing sequences may include self-splicing replacement intron-exon sequences derived from T4 phage gene td, and the tetrahymena intervening sequence (INTERVENING SEQUENCE, IVS) rRNA, the anabaena (cyanobacterium Anabaena) pre-tRNA-Leu gene, or the tetrahymena pre-rRNA.

In some embodiments, the polyribonucleotide comprises a catalytic intron fragment, e.g., the 3 'half of a type I catalytic intron fragment and the 5' half of a type I catalytic intron fragment. The first and second annealing regions can be located within the catalytic intron fragment. Type I catalytic introns are self-splicing ribozymes that catalyze their excision from mRNA, tRNA, and rRNA precursors by a bimetallic ion phosphoryl transfer mechanism. Importantly, the RNA itself self catalyzes the removal of introns without the need for exogenous enzymes such as ligases.

In some embodiments, the 3 'half of the type I catalytic intron fragment and the 5' half of the type I catalytic intron fragment are from the anabaena pre-tRNA-Leu gene, or Tetrahymena pre-rRNA.

In some embodiments, the 3 'half of the type I catalytic intron fragment and the 5' half of the type I catalytic intron fragment are from the anabaena pre-tRNA-Leu gene, and the 3 'exon fragment comprises a first annealing region and the 5' exon fragment comprises a second annealing region. The first annealing region may comprise, for example, 5 to 50, such as 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides, and the second annealing region may comprise, for example, 5 to 50, such as 10 to 15 (e.g., 10, 11, 12, 13, 14, or 15) ribonucleotides.

In some embodiments, the 3 'half of the group I catalytic intron fragment and the 5' half of the type I catalytic intron fragment are from tetrahymena pre-rRNA, and the 3 'half of the type I catalytic intron fragment comprises a first annealing region and the 5' exon fragment comprises a second annealing region. In some embodiments, the 3 'exon comprises a first annealing region and the 5' half of the type I catalytic intron fragment comprises a second annealing region. The first annealing region may comprise, for example, 6 to 50, such as 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, 6 to 50, such as 10 to 16 (e.g., 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

In some embodiments, the 3 'half of the type I catalytic intron fragment and the 5' half of the type I catalytic intron fragment are from the anabaena pre-tRNA-Leu gene, the tetrahymena pre-rRNA, or the T4 phage td gene.

In some embodiments, the 3 'half of the type I catalytic intron fragment and the 5' half of the type I catalytic intron fragment are from the T4 bacteriophage td gene. The 3 'exon fragment may comprise a first annealing region and the 5' half of the type I catalytic intron fragment may comprise a second annealing region. The first annealing region may comprise, for example, from 2 to 16, such as from 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, from 2 to 16, such as from 10 to 16 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

In some embodiments, the type I catalytic intron fragment is from the T4 phage nrdB gene or nrdD gene. In some embodiments, the 5' half of the type I catalytic intron fragment is from the T4 phage nrdB gene. In some embodiments, the 3 'half of the type I catalytic intron fragment is from the T4 phage nrdB gene and the 5' half of the type I catalytic intron fragment is from the T4 phage nrdB gene. In some embodiments, the 5' half of the type I catalytic intron fragment is from the T4 phage nrdD gene. In some embodiments, the 3 'half of the type I catalytic intron fragment is from the T4 phage nrdD gene and the 5' half of the type I catalytic intron fragment is from the T4 phage nrdD gene.

The 3 'exon fragment may comprise a first annealing region and the 5' half of the type I catalytic intron fragment may comprise a second annealing region. The first annealing region may comprise, for example, from 2 to 16, such as from 10 to 16 (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides, and the second annealing region may comprise, for example, from 2 to 16, such as from 10 to 16 (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16) ribonucleotides.

In some embodiments, the 3 'half of the type I catalytic intron fragment is the 5' end of the linear polynucleotide.

In some embodiments, the 5 'half of the type I catalytic intron fragment is the 3' end of a linear polyribonucleotide.

In another example, the linear polyribonucleotide may be circularized or interlinked by a non-nucleic acid moiety that results in attraction between the surfaces of atoms, molecules located at the 5 'and 3' ends of the linear polyribonucleotide, between the surfaces of atoms, molecules near the 5 'and 3' ends of the linear polyribonucleotide, or between the surfaces of atoms, molecules attached to the 5 'and 3' ends of the linear polyribonucleotide. One or more linear polyribonucleotides may be cyclized or interlinked by intermolecular forces or intramolecular forces. Non-limiting examples of intermolecular forces include dipole-dipole forces, dipole-induced dipole forces, induced dipole-induced dipole forces, van der waals forces, and london dispersion forces. Non-limiting examples of intramolecular forces include covalent bonds, metallic bonds, ionic bonds, resonance bonds, hydrogen-grasping bonds (diagnostic bonds), dipole bonds, conjugation, super-conjugation, and counter-bonds.

In another example, the linear polyribonucleotide may comprise a ribozyme RNA sequence near the 5 'end and near the 3' end. When the ribozyme RNA sequence is exposed to the remainder of the ribozyme, the sequence can be covalently linked to the peptide. Peptides covalently linked to ribozyme RNA sequences near the 5 'and 3' ends can bind to each other, resulting in linear polyribonucleotide cyclization or concatemerization. In another example, peptides covalently linked to ribozyme RNA near the 5 'and 3' ends can result in cyclization or concatemerization of a linear primary construct or linear mRNA after ligation using a variety of methods known in the art (e.g., without limitation, protein ligation). A non-limiting example of a ribozyme or method of incorporating or covalently linking a peptide for use in the linear primary construct or linear polyribonucleotide of the present invention is described in U.S. patent publication No. US20030082768 (the contents of which are incorporated herein by reference in their entirety).

Methods of making the circular polyribonucleotides described herein are described in, for example, Khudyakov&Fields, Artificial DNA: Methods and Applications, CRC Press (2002);Zhao, Synthetic Biology: Tools and Applications,（ first edition), ACADEMIC PRESS (2013), muller and Appel, from RNA Biol, 2017, 14 (8): 1018-1027, and Egli & Herdewijn, CHEMISTRY AND Biology of Artificial Nucleic Acids (first edition), wiley-VCH (2012). Other methods of preparing circular polyribonucleotides are described in, for example, international publication No. WO2023/044006, international publication No. WO2022/247943, U.S. Pat. No. US11000547, international publication No. WO2018/191722, international publication No. WO2019/236673, international publication No. WO2020/023595, international publication No. WO2022/204460, international publication No. WO2022/204464, international publication No. WO2022/204466, and International publication No. 2022/261490, each of which are incorporated herein by reference in their entirety.

Other methods of synthesizing cyclic polyribonucleotides are also described elsewhere (see, e.g., U.S. patent No. US6210931, U.S. patent No. US5773244, U.S. patent No. US5766903, U.S. patent No. US5712128, U.S. patent No. US5426180, U.S. publication No. US20100137407, international publication No. WO1992001813, international publication nos. WO2010084371 and Petkovic et al, nucleic Acids res.43:2454-65 (2015), the contents of each of which are incorporated herein by reference in their entirety).

In some embodiments, purified cyclic polyribonucleotides, e.g., free ribonucleic acids, linear or nicked RNAs, DNA, proteins, and the like are removed. In some embodiments, the cyclic polyribonucleotides can be purified by any known method commonly used in the art. Examples of non-limiting purification methods include column chromatography, gel excision, size exclusion, and the like.

Method of production in cell-free systems

In some embodiments, the circular polyribonucleotides described herein can be produced by transcription of a deoxyribonucleotide template in a cell-free system (e.g., by in vitro transcription) to produce linear polyribonucleotides. Linear polyribonucleotides produce splice compatible polyribonucleotides that can be self-spliced to produce cyclic polyribonucleotides.

In some embodiments, the cyclic polyribonucleotides are produced (e.g., in a cell-free system) by providing a linear polyribonucleotide, and self-splicing the linear polyribonucleotide under conditions suitable for splicing the 3 'and 5' splice sites of the linear polyribonucleotide, thereby producing the cyclic polyribonucleotide.

In some embodiments, the cyclic polyribonucleotides are produced by providing deoxyribonucleotides encoding the linear polyribonucleotides, transcribing the deoxyribonucleotides in a cell-free system to produce linear polyribonucleotides, optionally purifying splice-compatible linear polyribonucleotides, and self-splicing the linear polyribonucleotides under conditions suitable for splicing the 3 'and 5' splice sites of the linear polyribonucleotides to produce the cyclic polyribonucleotides.

In some embodiments, the cyclic polyribonucleotides are produced by providing deoxyribonucleotides encoding linear polyribonucleotides, and transcribing the deoxyribonucleotides in a cell-free system to produce linear polyribonucleotides, wherein the transcription is performed in solution under conditions suitable for splicing the 3 'and 5' splice sites of the linear polyribonucleotides, thereby producing the cyclic polyribonucleotides. In some embodiments, the linear polyribonucleotide comprises a 5 'splice intron and a 3' splice intron (e.g., a self-splice construct for producing a cyclic polyribonucleotide). In some embodiments, the linear polyribonucleotide comprises a 5 'annealing region and a 3' annealing region.

Suitable conditions for in vitro transcription and/or self-splicing may include any condition (e.g., a solution or buffer, e.g., an aqueous buffer or solution) that mimics a physiological condition in one or more respects. In some embodiments, suitable conditions include 0.1 to 100mM Mg ²⁺ ions or salts thereof (e.g., 1 to 100mM, 1 to 50mM, 1 to 20 mM, 5 to 50mM, 5 to 20 mM, or 5 to 15 mM). In some embodiments, suitable conditions include 1-1000 mM K ⁺ ions or salts thereof, such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 1-1000 mM Cl ^- ions or salts thereof, such as KCl (e.g., 1-1000 mM, 1-500 mM, 1-200 mM, 50-500 mM, 100-500 mM, or 100-300 mM). In some embodiments, suitable conditions include 0.1-100mM Mn ²⁺ ion or a salt thereof, such as MnCl ₂ (e.g., ,0.1-100 mM、0.1-50 mM、0.1-20 mM、0.1-10 mM、0.1-5 mM、0.1-2 mM、0.5-50 mM、0.5-20 mM、0.5-15 mM、0.5-5 mM、0.5-2 mM or 0.1-10 mM). In some embodiments, suitable conditions include Dithiothreitol (DTT) (e.g., ,1-1000 μM、1-500 μM、1-200 μM、50-500 μM、100-500 μM、100-300 μM、0.1-100 mM、0.1-50 mM、0.1-20 mM、0.1-10 mM、0.1-5 mM、0.1-2 mM、0.5-50 mM、0.5-20 mM、0.5-15 mM、0.5-5 mM、0.5-2 mM or 0.1-10 mM). In some embodiments, suitable conditions include 0.1 mM to 100mM ribonucleoside triphosphates (NTPs) (e.g., 0.1-100mM, 0.1-50 mM, 0.1-10 mM, 1-100mM, 1-50 mM, or 1-10 mM). In some embodiments, suitable conditions include a pH of 4 to 10 (e.g., pH 5 to 9, pH 6 to 9, or pH 6.5 to 8.5). In some embodiments, suitable conditions include a temperature of 4 ℃ to 50 ℃ (e.g., 10 ℃ to 40 ℃, 15 ℃ to 40 ℃,20 ℃ to 40 ℃, or 30 ℃ to 40 ℃).

In some embodiments, the linear polyribonucleotides are produced from deoxyribonucleic acids, such as those described herein, e.g., DNA vectors, linearized DNA vectors, or cdnas. In some embodiments, the linear polyribonucleotides are transcribed from deoxyribonucleic acid (e.g., in vitro transcription) by transcription in a cell-free system.

Method of production in cells

In some embodiments, the cyclic polyribonucleotide is produced in a cell, such as a prokaryotic cell or eukaryotic cell. In some embodiments, the cell is provided with an exogenous polyribonucleotide (e.g., a linear polyribonucleotide or a DNA molecule encoding transcription of a linear polyribonucleotide). The linear polyribonucleotides can be transcribed in the cell from an exogenous DNA molecule provided to the cell. The linear polyribonucleotides can be transcribed in a cell from an exogenous recombinant DNA molecule that is transiently supplied to the cell. In one embodiment, the linear polyribonucleotides can be transcribed in the cell from an exogenous DNA molecule provided to the cell (e.g., with a plasmid). In some embodiments, the exogenous DNA molecule is not integrated into the genome of the cell. In some embodiments, the linear polyribonucleotides are transcribed in the cell from a recombinant DNA molecule that is incorporated into the genome of the cell.

In some embodiments, the cell is a prokaryotic cell. In some embodiments, the prokaryotic cell comprising the polyribonucleotides described herein may be a bacterial cell or an archaeal cell. For example, a prokaryotic cell comprising a polyribonucleotide described herein can be escherichia coli, halophilus (halophilic archaea) (e.g., volvulus halophilus (Haloferax volcaniii)), sphingomonas (sphangomonas), cyanobacteria (cyanobacteria) (e.g., synechococcus (Synechococcus elongatus), spirulina (spira sp.) (arthrospira (Arthrospira sp.)) and Synechocystis (Synechocystis sp.)), streptomyces (Streptomyces), actinomycetes (e.g., wild-type bacteria (Nonomuraea), north-spore (Kitasatospora) or thermomyces (Thermobifida)), bacillus (Bacillus sp.) (e.g., bacillus subtilis (Bacillus subtilis), bacillus anthracis (Bacillus anthracis), bacillus cereus (Bacillus)), beta-proteus (betaproteobacteria) (e.g., burkholderia (e.g., pseudomonas (α)), pseudomonas (32) (e.g., pseudomonas (32) and Pseudomonas (Pseudomonas putida)), and Pseudomonas (Pseudomonas putida). Prokaryotic cells may be grown in culture. The prokaryotic cells may be contained in a bioreactor.

In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell comprising a polyribonucleotide described herein is a single cell eukaryotic cell. In some embodiments, the unicellular eukaryotic organism is a unicellular fungal cell, such as a yeast cell (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae) and other Saccharomyces (Saccharomyces spp.), brettanomyces spp.), schizosaccharomyces (Schizosaccharomyces spp.), torulopsis (torula spp.), and Pichia spp.). In some embodiments, the single cell eukaryotic cell is a single cell animal cell. The single cell animal cell may be a cell isolated from a multicellular animal and grown in culture, or a daughter cell thereof. In some embodiments, single cell animal cells may be dedifferentiated. In some embodiments, the single cell eukaryotic cell is a single cell plant cell. The single-cell plant cell may be a cell isolated from a multicellular plant and grown in culture, or a daughter cell thereof. In some embodiments, single cell plant cells may be dedifferentiated. In some embodiments, the single cell plant cell is from a plant callus. In embodiments, the single cell is a plant cell protoplast. In some embodiments, the single cell eukaryotic cell is a single cell eukaryotic algal cell, such as a single cell green algae, diatom, euglena, or dinoflagellate. Non-limiting examples of unicellular eukaryotic algae of interest include dunaliella salina (Dunaliella salina), chlorella vulgaris (Chlorella vulgaris), zofeno chlorella (Chlorella zofingiensis), rhodococcus pluvialis (Haematococcus pluvialis), neo-green algae rich in oil (Neochloris oleoabundans) and other neo-green algae (Neochloris spp.), prototheca botryoides (Protosiphon botryoides), botryococcus braunii (Botryococcus braunii), cryptococcus (Cryptococcus spp.), chlamydomonas reinhardtii (Chlamydomonas reinhardtii) and other Chlamydomonas spp. In some embodiments, the single cell eukaryotic cell is a protist cell. In some embodiments, the single cell eukaryotic cell is a protozoan cell.

In some embodiments, the eukaryotic cell is a multicellular eukaryotic cell. For example, the multicellular eukaryotic organism may be selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular algae, and multicellular plants. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryote is an invertebrate. In some embodiments, the eukaryote is a multicellular fungus. In some embodiments, the eukaryote is a multicellular plant. In embodiments, the eukaryotic cells are cells of a human or non-human mammal, such as a non-human primate (e.g., monkey, ape), ungulate (e.g., bovine, including bovine (cattle), buffalo (buffalo), bison (bison), sheep, goat, and musk; swine; camel, including camel, llama, and alpaca; deer, antelope; and equine, including horse and donkey), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or rabbit (e.g., rabbit, hare). In embodiments, the eukaryotic cell is a cell of a bird, which is, for example, a member of the avian classification galliformes (e.g., chicken, turkey, pheasant, quail), anseriformes (e.g., duck, goose), gullet (Paleaognathae) (e.g., ostrich, emu), pigeon (e.g., pigeon, vernonia) or psittacosis (e.g., parrot). In embodiments, the eukaryotic cell is a cell of an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, helminth, or mollusc. In embodiments, the eukaryotic cell is a cell of a multicellular plant, such as an angiosperm (which may be a dicotyledonous plant or a monocotyledonous plant) or a gymnosperm (e.g., conifer, cymbidium, gnetitum, ginkgo), fern, horsetail, pinus koraiensis, or bryophyte. In embodiments, the eukaryotic cell is a cell of a eukaryotic multicellular algae.

Eukaryotic cells may be grown in culture. Eukaryotic cells may be contained in the bioreactor.

Purification method

One or more purification steps may be included in the methods described herein. For example, in some embodiments, the linear polyribonucleotides are substantially enriched or pure (e.g., purified) prior to self-splicing the linear polyribonucleotides. In other embodiments, the linear polyribonucleotides are not purified prior to self-splicing the linear polyribonucleotides. In some embodiments, the resulting cyclic polyribonucleotides are purified.

Purification may include separation or enrichment of the desired reaction product from one or more undesired components (e.g., any unreacted starting materials, byproducts, enzymes, or other reaction components). For example, purifying linear polyribonucleotides after transcription (e.g., in vitro transcription) in a cell-free system can include isolation or enrichment from a DNA template prior to self-splicing of the linear polyribonucleotides. Purification of the cyclic polyribonucleotide product after splicing can be used to separate or enrich the cyclic polyribonucleotides from their corresponding linear polyribonucleotides. Methods of purification of RNA are known to those skilled in the art and include enzyme purification or purification by chromatography.

In some embodiments, the purification method produces cyclic polyribonucleotides with less than 50% (e.g., less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%) of linear polyribonucleotides.

Bioreactor

In some embodiments, any method of producing a cyclic polyribonucleotide described herein can be performed in a bioreactor. A bioreactor refers to any container in which a chemical or biological process involving an organism or a biochemically active substance derived from such an organism is performed. The bioreactor may be compatible with cell-free methods for producing the cyclic polyribonucleotides described herein. The container of the bioreactor may comprise a culture flask, a culture dish or a bag, which may be single-use (disposable), autoclavable or sterilizable. The bioreactor may be made of glass, or it may be a polymer-based material, or it may be made of other materials.

Examples of bioreactors include, but are not limited to, stirred tank (e.g., well-mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibratory mixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of the bioreactor may be a batch or continuous process. The bioreactor is continuous as the reagent and product streams are continuously fed to and withdrawn from the system. The batch bioreactor may have a continuous recycle stream, but no continuous feed of reagents or continuous collection of products.

Some methods of the present disclosure are directed to mass production of cyclic polyribonucleotides. For large scale production processes, the process can be performed in a volume of 1 liter (L) to 50L or more (e.g., 5L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, 50L or more). In some embodiments, the method may be performed in a volume of 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15 to 50L.

In some embodiments, the bioreactor may produce at least 1g cyclic polyribonucleotides. In some embodiments, the bioreactor can produce 1-200 g loop polyribonucleotides (e.g., 1-10 g, 1-20 g, 1-50 g, 10-50 g, 10-100 g, 50-100 g, or 50-200 g loop RNA). In some embodiments, the throughput is measured per liter (e.g., 1-200 g per liter), per batch or per reaction (e.g., 1-200 g per batch or per reaction), or per unit time (e.g., 1-200 g per hour or day).

In some embodiments, more than one bioreactor may be utilized in series to increase production capacity (e.g., one, two, three, four, five, six, seven, eight, or nine bioreactors may be utilized in series).

Gene editing

In some cases, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can include components of a gene editing system. For example, an agent may introduce a change (e.g., an insertion, a deletion (e.g., a knockout), a translocation, an inversion, a single point mutation, or other mutation) in a gene in a target organism. Exemplary gene editing systems include Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Methods based on ZFN, TALEN and CRISPR are described, for example, in Gaj et al, trends biotechnol.31 (7): 397-405, 2013.

Additional description of the components and processes of the gene editing system can be found in International patent application publication No. WO 2021/04301 (the entire contents of which are incorporated herein by reference).

MRNA therapeutic agent and circRNA therapeutic agent

The LNMP/mRNA composition comprises one or more polynucleotides (e.g., mRNA) encoding one or more antigenic polypeptides or signaling polypeptides for therapeutic purposes (e.g., against cancer). The one or more polynucleotides (e.g., mRNA) encode one or more cancer (tumor) antigen polypeptides. In some embodiments, the LNMP/circRNA therapeutic composition comprises one or more cyclic polyribonucleotides. In some embodiments, a cyclic polyribonucleotide includes one or more polyribonucleotides (e.g., one or more polyribonucleotide cargo of the polyribonucleotide) that encodes one or more antigen polypeptides or signaling polypeptides for therapeutic purposes (e.g., against cancer). In some embodiments, the polyribonucleotides include one or more expression sequences encoding one or more antigen (e.g., tumor antigen) polypeptides or signaling polypeptides.

Tumor antigens

Cancers or tumors include, but are not limited to, neoplasms, malignant tumors, metastases, or any disease or disorder in which cell growth is uncontrolled and considered cancerous. The cancer may be a primary cancer or a metastatic cancer. Specific cancers that may be treated according to the present invention include, but are not limited to, the cancers listed below (for a review of such disorders, see FISHMAN ET AL, 1985, medicine, 2d Ed., j.b. Lippincott co., philia). Cancers include, but are not limited to, biliary tract cancer, bladder cancer, brain cancer (including glioblastoma and medulloblastoma), breast cancer, cervical cancer, choriocarcinoma, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms (including acute lymphoblastic leukemia and acute myeloid leukemia), multiple myeloma, AIDS-related leukemia and adult T-cell leukemia lymphomas, intraepithelial neoplasms (including Bowen's disease and Paget's disease), liver cancer, lung cancer, lymphomas (including Hodgkin's disease and lymphoblastic lymphoma), neuroblastoma, oral cancer (including squamous cell carcinoma), ovarian cancer (including ovarian cancer caused by epithelial cells, stromal cells, germ cells and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma), skin cancers (including melanoma, kaposi's (Kaposi's sarcoma), basal cell carcinoma, testicular cancer (including germ cell carcinoma), germ cell carcinoma (including germ cell carcinoma), wilm's cell carcinoma, wilm's cancer, and carcinoma, thyroid cancer (including carcinoma), thyroid cancer, and carcinoma, and wilm's cancer, and carcinoma. Common cancers include breast cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, and brain cancer.

In some embodiments, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), small cell lung cancer, melanoma, bladder urothelial cancer, HPV negative Head and Neck Squamous Cell Carcinoma (HNSCC), and microsatellite high (MSI H)/mismatch repair (MMR) deficient solid malignancies. In some embodiments, NSCLC lacks EGFR priming mutations and/or ALK translocation. In some embodiments, the microsatellite high (MSI H)/mismatch repair (MMR) deficient solid malignancy is selected from the group consisting of colorectal cancer, gastric adenocarcinoma, esophageal adenocarcinoma, and endometrial cancer. In some embodiments, the cancer is selected from pancreatic cancer, peritoneal cancer, colorectal cancer, small intestine cancer, biliary tract cancer, lung cancer, endometrial cancer, ovarian cancer, genital tract cancer, gastrointestinal cancer, cervical cancer, gastric cancer, urinary tract cancer, colon cancer, rectal cancer, hematopoietic and lymphoid tissue cancer. In some embodiments, the cancer is colorectal cancer.

In some embodiments, the tumor antigen polypeptide comprises p53, ART-4, BAGE, ss-catenin /m、Bcr-abL CAMEL、CAP-1、CASP-8、CDC27/m、CDK4/m、CEA、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, MC R, myosin/m, MUC1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 times BCR-abL, plac-1, pml/RARa, PRE, protease 3, PSA, PSM, RAGE, RU1 or 2, SAGE, SART-1 or SART-A2, SART-3, SCL-A3, SCP-2, SCP-3/TRP-2, TRP-3, TRP-57, TRP-2, or a combination thereof.

In some embodiments, the tumor antigen is one of ：CD2、CD3、CD4、CD8、CD11b、CD14、CD16、CD19、CD20、CD22、CD25、CD27、CD33、CD37、CD38、CD40、CD44、CD45、CD47、CD52、CD56、CD70、CD79、CD137、4-IBB、5T4、AGS-5、AGS-16、 angiopoietin 2, B7.1, B7.2, B7DC, B7H1, B7H2, B7H3, BT-062, BTLA, CAIX, carcinoembryonic antigen, CTLA4, cripto, ED-B, erbBl, erbB2, erbB3, erbB4, EGFL7, epCAM, ephA2, ephA3, ephB2, FAP, fibronectin, folate receptor, ganglioside GM3, GD2, glucocorticoid-induced tumor necrosis factor receptor (GITR), gplOO, gpA33, GPNMB, HLA, HLA-DR, ICOS, IGF R, integrin av, integrin αvβ, G-3, lewis Y, mesothelin, c-MET, MN carbonic anhydrase IX, MUC1, MUC16, fibronectin-4, KGD2, NOTCH, OX40L, PD-1, PDL1, OX PSCA, PSMA, RANKL, ROR, ROR 4, SLC-4, LAFR-42, tenascin, TAG 3, and variants thereof.

In some embodiments, the tumor antigen polypeptide is an IL2 peptide, IL-2-Ra, anti-CD 19 antibody, anti-CD 20 antibody, chimeric antigen receptor T cell (CAR-T) antibody, anti-HER 2 antibody, etanercept (etanercept) (e.g., enbrel), adalimumab (e.g., semanteb (Humira)), epoetin alfa (e.g., epogen), feglastin (filgrastim) (e.g., neupogen), pembrolizumab (e.g., keytruda (Keytruda)), rituximab (rituximab) (e.g., rituximab (Rituxan)), romidepsin (romiplostim) (e.g., nplate), saxapavilion (sargramostim) (e.g., leukine), or a fragment or subunit thereof. In one embodiment, the tumor antigen polypeptide is an IL2 peptide, or a fragment or subunit thereof. In one embodiment, the tumor antigen polypeptide is epoetin (epoetin alfa) (e.g., epogen), or a fragment or subunit thereof.

In some embodiments, the tumor antigen polypeptide comprises a tumor antigen selected from the group consisting of carcinoma, sarcoma, melanoma, lymphoma, leukemia, and combinations thereof.

In one embodiment, the tumor antigen polypeptide comprises a melanoma tumor antigen. In one embodiment, the tumor antigen polypeptide comprises a prostate cancer antigen. In one embodiment, the tumor antigen polypeptide comprises HPV 16-positive head and neck cancer antigen. In one embodiment, the tumor antigen polypeptide comprises a breast cancer antigen. In one embodiment, the tumor antigen polypeptide comprises an ovarian cancer antigen. In one embodiment, the tumor antigen polypeptide comprises a lung cancer antigen. In one embodiment, the tumor antigen polypeptide comprises an NSCLC antigen.

In some embodiments, the antigen is an autoantigen polypeptide or an immunogenic variant or immunogenic fragment thereof. In some embodiments, the autoantigen polypeptide comprises an antigen that is normally expressed on a cell and recognized by the immune system as an autoantigen. In some embodiments, the autoantigen polypeptide comprises a multiple sclerosis antigen polypeptide, a rheumatoid arthritis antigen polypeptide, a lupus antigen polypeptide, a diarrhea celiac disease antigen polypeptide, a Sjogren's syndrome antigen polypeptide, or a ankylosing spondylitis antigen polypeptide, or a combination thereof.

Immunopotentiator mRNA

In some embodiments, the one or more polynucleotides comprise mRNA encoding a polypeptide that stimulates or enhances an immune response against the one or more cancer antigens of interest. Such mRNA that enhances an immune response to a cancer antigen of interest is referred to herein as an immunopotentiator mRNA construct or immunopotentiator mRNA, including chemically modified mRNA (mmRNA). In some embodiments, the cyclic polyribonucleotides disclosed herein comprise one or more expression sequences encoding polypeptides that stimulate or enhance an immune response against one or more cancer antigens of interest. Such cyclic polyribonucleotides that enhance the immune response to a cancer antigen of interest are referred to herein as immunopotentiator circRNA. The immunopotentiator enhances an immune response to the antigen of interest in the subject. The enhanced immune response may be a cellular response, a humoral response, or both. As used herein, "cellular" immune response is intended to include immune responses involving or mediated by T cells, while "humoral" immune response is intended to include immune responses involving or mediated by B cells. Immunopotentiators can enhance immune responses by, for example:

(i) Stimulating type I interferon pathway signaling;

(ii) Stimulating NFkB pathway signaling;

(iii) Stimulating an inflammatory response;

(iv) Stimulating cytokine production, or

(V) Stimulating dendritic cell development, activity or mobilization, and

(Vi) A combination of any of (i) - (vi).

As used herein, "stimulating type I interferon pathway signaling" is intended to include activating one or more components of a type I interferon signaling pathway (e.g., modifying phosphorylation, dimerization, etc. of such components to activate the pathway), stimulating transcription of an Interferon Sensitive Response Element (ISRE), and/or stimulating production or secretion of type I interferon (e.g., IFN-a, IFN- β, IFN-epsilon, IFN-K, and/or IFN-co). As used herein, "stimulating NFkB pathway signaling" is intended to include activating one or more components of the NFkB signaling pathway (e.g., modifying phosphorylation, dimerization, etc. of such components to activate the pathway), stimulating transcription of the NFkB site, and/or stimulating production of a gene product that is expressed by NFkB regulation. As used herein, "stimulating an inflammatory response" is intended to include stimulating the production of inflammatory cytokines (including, but not limited to, type I interferons, IL-6, and/or TNFa). As used herein, "stimulating dendritic cell development, activity, or mobilization" is intended to include direct or indirect stimulation of dendritic cell maturation, proliferation, and/or functional activity.

In some embodiments, the mRNA or circRNA encodes a polypeptide that stimulates or enhances an immune response in a subject in need thereof (e.g., enhances an immune response in a subject) by, for example, inducing adaptive immunity in a subject (e.g., by stimulating type I interferon production), stimulating an inflammatory response, stimulating FkB signaling, and/or stimulating the development, activity, or mobilization of Dendritic Cells (DCs). In some embodiments, administration of an immunopotentiator mRNA or circRNA to a subject in need thereof enhances cellular immunity (e.g., T cell mediated immunity), humoral immunity (e.g., B cell mediated immunity), or both cellular and humoral immunity of the subject. In some embodiments, administration of the immunopotentiator mRNA or circRNA stimulates cytokine production (e.g., inflammatory cytokine production), stimulates a cancer antigen specific CD8 ⁺ effector cell response, stimulates an antigen specific CD4 ⁺ helper cell response, increases effector memory CD62L ¹⁰ T cell populations, stimulates B cell activity, or stimulates production of antigen specific antibodies, including combinations of the foregoing. In some embodiments, administration of the immunopotentiator mRNA or circRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD8 ⁺ effector cell responses. In some embodiments, administration of the immunopotentiator mRNA or circRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates antigen-specific CD4 ⁺ to assist in the cellular response. In some embodiments, administration of the immunopotentiator mRNA or circRNA stimulates cytokine production (e.g., inflammatory cytokine production) and increases the effector memory CD62L ¹⁰ T cell population. In some embodiments, administration of the immunopotentiator mRNA or circRNA stimulates cytokine production (e.g., inflammatory cytokine production) and stimulates B cell activity or stimulates production of antigen-specific antibodies.

In one embodiment, the immunopotentiator increases the cancer antigen specific CD8 ⁺ effector cell response (cellular immunity). For example, an immunopotentiator may increase one or more indicators of antigen-specific CD8 ⁺ effector cell activity, including, but not limited to, CD8 ⁺ T cell proliferation and CD8 ⁺ T cytokine production. for example, in one embodiment, the immunopotentiator increases antigen-specific CD8 ⁺ T cells producing IFN-gamma, TNFa and/or IL-2. In various embodiments, the immunopotentiator may increase CD8 ⁺ T cytokine production (e.g., IFN- γ, TNFa and/or IL-2 production) by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30%) in response to an antigen (as compared to CD8 ⁺ T cytokine production in the absence of the immunopotentiator), or at least 35% or at least 40% or at least 45% or at least 50%. For example, T cells obtained from a treated subject can be stimulated in vitro with a cancer antigen, and the production of CD8 ⁺ T cell cytokines can be assessed in vitro. Production of CD8 ⁺ T-cell cytokines can be determined by standard methods known in the art, including, but not limited to, measuring secreted cytokine production levels (e.g., by ELISA or other suitable methods known in the art for determining the amount of cytokine in the supernatant) and/or determining the percentage of CD8 ⁺ T-cells positive for intracellular staining (ICS) of the cytokine. For example, CD8 ⁺ T cells can be subjected to intracellular staining (ICS) for IFN-gamma, TNFa and/or JL-2 expression by methods known in the art. in one embodiment, the immunopotentiator responds to the antigen by directing the antigen through the ICS towards one or more cytokines (e.g., IFN-gamma, TNFa and/or IL-2) increases the percentage of CD8 ⁺ T cells positive (compared to the percentage of CD8 ⁺ T cells positive for the cytokine by ICS in the absence of immunopotentiator) by at least 5% or at least 10% or at least 15% >, or at least 20% or at least 25% or at least 30% >, or at least 35% or at least 40% or at least 45% or at least 50%.

In one embodiment, the immunopotentiator increases the percentage of CD8 ⁺ T cells in the total T cell population (e.g., splenic T cells and/or PBMCs) compared to the percentage of CD8 ⁺ T cells in the absence of the immunopotentiator. For example, an immunopotentiator may increase the percentage of CD8 ⁺ T cells in the total T cell population by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30%) or at least 35% or at least 40% or at least 45% or at least 50% as compared to the percentage of CD8 ⁺ T cells in the absence of the immunopotentiator. The total percentage of CD8 ⁺ T cells in the total T cell population can be determined by standard methods known in the art, including, but not limited to, fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS).

In one embodiment, the immunopotentiator increases a tumor-specific immune cell response as determined by the decrease in tumor volume in vivo in the presence of the immunopotentiator as compared to the tumor volume in the absence of the immunopotentiator. For example, an immunopotentiator may reduce the tumor volume by at least 5% or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, compared to the tumor volume in the absence of the immunopotentiator. The measurement of tumor volume can be determined by methods well known in the art. In another embodiment, the immunopotentiator increases B cell activity (humoral immune response), for example by increasing the amount of antigen-specific antibody produced, as compared to antigen-specific antibody produced in the absence of the immunopotentiator. For example, an immunopotentiator may increase antigen-specific antibody production by at least 5% or at least 10% or at least 15% or at least 20% or at least 25%) or at least 30% >, or at least 35% or at least 40% or at least 45% or at least 50% as compared to antigen-specific antibody production in the absence of the immunopotentiator. In one embodiment, the production of antigen-specific IgG is assessed. The production of antigen-specific antibodies can be assessed by well-established methods in the art, including but not limited to ELISA, RIA, etc., which measure the level of antigen-specific antibodies (e.g., igG) in a sample (e.g., serum sample).

In one embodiment, the immunopotentiator increases the population of effector memory CD62L ¹⁰ T cells. For example, an immunopotentiator may increase the total% of CD62L ¹⁰ T cells in CD8 ⁺ T cells. Among other functions, effector memory CD62L ¹⁰ T cell populations have been shown to play an important role in lymphocyte transport (see, e.g., schenkel, j.m. and Masopust, d. (2014) Immunity 41:886-897). In various embodiments, the immunopotentiator may increase the total percentage of effector memory CD62L ¹⁰ T cells in CD8 ⁺ T cells (as compared to the total percentage of CD62L ¹⁰ T cells in the population of CD8 ⁺ T cells in the absence of the immunopotentiator) by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50% in response to the antigen. The total percentage of effector memory CD62L ¹⁰ T cells in CD8 ⁺ T cells can be determined by standard methods known in the art, including, but not limited to, fluorescence Activated Cell Sorting (FACS) or Magnetic Activated Cell Sorting (MACS).

The ability of the immunopotentiator mRNA or circRNA to enhance an immune response to a cancer antigen can be assessed in a mouse model system known in the art. In one embodiment, a mouse model system with immune competence is used. In one embodiment, the mouse model system comprises C57/B16 mice. In another embodiment, the mouse model system comprises a BalbC mouse or CD1 mouse { e.g., to assess B cell responses, such as antigen-specific antibody responses). In one embodiment, the immunopotentiator polypeptide acts downstream of at least one Toll-like receptor (TLR), thereby enhancing the immune response. Thus, in one embodiment, the immunopotentiator is not a TLR, but a molecule within the TLR signaling pathway downstream of the receptor itself.

In one embodiment, the mRNA or circRNA encoding the immunopotentiator may comprise one or more modified nucleobases. Suitable modifications are discussed further below.

In one embodiment, mRNA or circRNA encoding an immunopotentiator is formulated into an LNMP formulation. In one embodiment, the mRNA or circRNA encodes a cancer antigen. In one embodiment, the LNMP/mRNA formulation or LNMP/circRNA formulation is administered to the subject to enhance the subject's immune response to the cancer antigen.

Immunopotentiator mRNA stimulating type I interferon

In some embodiments, the present disclosure provides immunopotentiator mRNA or circRNA encoding a polypeptide that stimulates or enhances an immune response against an antigen of interest by stimulating or enhancing type I interferon pathway signaling, thereby stimulating or enhancing type I Interferon (IFN) production.

Many components involved in type I IFN pathway signaling have been identified, including STING, interferon regulatory factors such as IRF1, IRF3, IRF5, IRF7, IRF8 and IRF9, TBK1, IKKi, myD88 and TRAM. Other components involved in type I IFN pathway signaling include TRAF3, TRAF6, IRAK-1, IRAK-4, TRIF, IPS-1, TLR-3, TLR-4, TLR-7, TLR-8, TLR-9, RIG-1, DAI and IFI16.

Thus, in one embodiment, the immunopotentiator mRNA or circRNA encodes any of the aforementioned components involved in type I IFN pathway signaling.

Agents promoting antigen presenting cells

In some embodiments, an LNMP/RNA composition (e.g., an mRNA composition or a circRNA composition) can be combined with an agent for promoting Antigen Presenting Cell (APC) production (e.g., by converting non-APCs to pseudo (pseudo) APCs). Antigen presentation is a key step in the initiation, amplification and persistence of immune responses. In this process, the antigen fragment passes through:

Major Histocompatibility Complex (MHC) or Human Leukocyte Antigen (HLA) is presented to T cells, driving antigen-specific immune responses. For immunoprophylaxis and therapy, enhancing this response is important to enhance efficacy. LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) can be designed or enhanced to drive efficient antigen presentation. One approach to enhance APC processing and presentation is to better target LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) to Antigen Presenting Cells (APCs). Another method involves activating APC with an immunostimulatory agent and/or component. Alternatively, the method for reprogramming non-APCs to APCs can be used with an LNMP/RNA composition (e.g., an mRNA composition or a circRNA composition). Importantly, most cells that ingest mRNA preparations and are targets for their therapeutic action are not APCs. Therefore, it would be advantageous to devise a method of converting these cells into APC. Provided herein are methods and pathways for delivering LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions), such as mRNA or circRNA vaccines, to cells while promoting conversion of non-APCs to APCs. In some embodiments, the mRNA or circRNA encoding the APC reprogramming molecule is contained in or co-administered with an LNMP/RNA composition (e.g., an mRNA composition or a circRNA composition).

As used herein, an APC reprogramming molecule is a molecule that promotes the conversion of non-APC cells to an APC-like phenotype. APC-like phenotype is a property that enables class II MHC processing. Thus, an APC cell having an APC-like phenotype is a cell having one or more exogenous molecules (APC reprogramming molecules) that has enhanced MHC class II processing capacity compared to the same cell without the one or more exogenous molecules. In some embodiments, the APC reprogramming molecule is CUT A (central modulator of MHC class II expression), chaperones such as CLIP, HLA-DO, HLA-DM, and the like (enhancers for loading antigen fragments into MHC class II) and/or costimulatory molecules such as CD40, CD80, CD86, and the like (enhancers of T cell antigen recognition and T cell activation).

CIITA proteins are transactivators that enhance MHC class II gene transcriptional activation by interacting with a set of conserved DNA binding proteins associated with a class II promoter region (STEIMLE ET al., 1993, cell 75:135-146). The transcriptional activation function of CIITA has been aligned to the amino terminal acid domain (amino acids 26-137). The nucleic acid molecule encodes a protein that interacts with CIITA, which is referred to herein as CIITA interacting protein 104 (also referred to herein as CIP 104). CITTA and CIP104 have both been shown to enhance transcription of MHC class II promoters and thus are useful as APC reprogramming molecules in the present invention. In some embodiments, the APC reprogramming molecule is full length CIITA, CIP104, or other related molecule or active fragment thereof, such as amino acids 26-137 of CIITA, or amino acids having at least 80% sequence identity thereto and maintaining enhanced MHC class II gene transcriptional activation.

In some embodiments, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can comprise a recall antigen, sometimes also referred to as a memory antigen. Recall antigens are antigens against which an individual has encountered and against which memory lymphocytes are already present in the body. In some embodiments, the recall antigen may be an infectious disease antigen that the individual may have encountered, such as an influenza antigen. Recall that the antigen helps to promote a stronger immune response.

The antigen or neoepitope (neoepitope) selected into the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) is typically a high affinity binding peptide. In some embodiments, the antigen or neoepitope binds to HLA proteins with greater affinity than the wild-type peptide. In some embodiments, the IC50 of the antigen or neoepitope is at least less than 5000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM, or less. In general, peptides with predicted IC50<50 nM are generally considered medium to high affinity binding peptides and will be selected to empirically test their affinities using biochemical assays of HLA binding.

The cancer antigen may be a personalized cancer antigen. The LNMP/RNA composition (e.g., mRNA composition or circRNA composition) can comprise RNA encoding one or more known tumor-specific cancer antigens or each subject-specific cancer antigen (referred to as a neoepitope or subject-specific epitope or antigen). A "subject-specific cancer antigen" is an antigen that has been identified as being expressed in a tumor of a particular patient. The subject-specific cancer antigen may or may not be commonly present in a tumor sample. Tumor-associated antigens that are not or are rarely expressed in non-cancerous cells, or whose expression in non-cancerous cells is substantially reduced compared to that in cancerous cells and induce an immune response after vaccination are referred to as neo-epitopes. Like tumor-associated antigens, neoepitopes are completely foreign to the body and therefore do not produce an immune response against healthy tissue or are masked by protective components of the immune system. In some embodiments, neoepitope-based LNMP/RNA compositions (e.g., mRNA compositions or circRNA compositions) are desirable because such vaccine formulations will maximize specificity for a patient-specific tumor. Mutation-derived neoepitopes may be caused by point mutations, non-synonymous mutations resulting in amino acid differences in the protein, read-through mutations, in which the stop codon is modified or deleted, resulting in translation of longer proteins with new tumor-specific sequences at the C-terminus, splice site mutations, resulting in inclusion of introns in mature mRNA, thus producing unique tumor-specific protein sequences, chromosomal rearrangements, resulting in chimeric proteins with tumor-specific sequences at the junction of 2 proteins (i.e., gene fusions), frame shift mutations or deletions, resulting in new open reading frames with new tumor-specific protein sequences, and translocations.

Thus, in some embodiments, the LNMP/RNA composition (e.g., mRNA composition or circRNA composition) comprises at least 1 cancer antigen, including one selected from the group consisting of frameshift mutations and recombinations or any other mutation described herein.

Nucleic acid sequences

In some embodiments, the polynucleotide is a polynucleotide construct that encodes one or more wild-type or engineered antigens (or antibodies to antigens). In some embodiments, the polynucleotide comprises a tumor antigen polypeptide or an immunogenic variant or immunogenic fragment thereof. The antigen may be derived from a tumor, such as a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof.

In some embodiments, the polynucleotide may be mRNA, siRNA or siRNA precursor, microRNA (miRNA) or miRNA precursor, plasmid, dicer substrate small interfering RNA (dsiRNA), short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), peptide Nucleic Acid (PNA), morpholino, locked Nucleic Acid (LNA), piwi interacting RNA (piRNA), ribozyme, deoxyribose enzyme (DNAzyme), aptamer, circular RNA (circRNA), guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is mRNA.

In some embodiments, the polyribonucleotide comprises a plurality of sequences, each sequence encoding an antigen, and at least one sequence of the plurality of sequences encodes a coronavirus antigen. In some embodiments, the circular polyribonucleotide comprises two or more ORFs. In some embodiments, the cyclic polyribonucleotides comprise at least five sequences, each sequence encoding one antigen, and at least one antigen is a coronavirus antigen. In some embodiments, the cyclic polyribonucleotide comprises at least two ORFs, e.g., at least 2,3, 4, or 5 ORFs. In some embodiments, the cyclic polyribonucleotides comprise 5 to 20 sequences, each sequence encoding one antigen, and at least one antigen is a coronavirus antigen. In some embodiments, the cyclic polyribonucleotides comprise 5 to 10 sequences, each sequence encoding one antigen, and at least one antigen is a coronavirus antigen. In some embodiments, the cyclic-polyribonucleotide comprises a sequence that encodes an antigen from at least two different microorganisms, and at least one microorganism is a coronavirus.

In some embodiments, the cyclic polyribonucleotides comprise one or more elements described herein in addition to a binding site (e.g., a sequence for binding to a target). In some embodiments, the cyclic polyribonucleotide lacks a poly-A tail. In some embodiments, the circular polyribonucleotide lacks a replicating element. In some embodiments, the circular polyribonucleotide lacks an IRES. In some embodiments, the cyclic polyribonucleotide lacks a cap. In some embodiments, the circular polyribonucleotides, in addition to the binding site, further comprise any feature or any combination of features disclosed in WO2019/118919 (the entire contents of which are incorporated herein by reference).

In some embodiments, the circRNA may comprise at least one binding site for a target (e.g., for a binding portion of the target). The circRNA may comprise at least one aptamer sequence that binds to a target. In some embodiments, the circRNA comprises one or more binding sites for one or more targets. Targets include, but are not limited to, nucleic acids (e.g., RNA, DNA, RNA-DNA hybrids), small molecules (e.g., drugs, fluorophores, metabolites), aptamers, polypeptides, proteins, lipids, carbohydrates, antibodies, viruses, viral particles, membranes, multicomponent complexes, organelles, cells, other cell parts, any fragments thereof, and any combination thereof. (see, e.g., fredriksson et al, (2002) Nat Biotech 20:473-77;Gullberg et al, (2004) PNAS, 101:8420-24). For example, the target is single-stranded RNA, double-stranded RNA, single-stranded DNA, double-stranded DNA, DNA or RNA comprising one or more double-stranded regions and one or more single-stranded regions, RNA-DNA hybrids, small molecules, aptamers, polypeptides, proteins, lipids, carbohydrates, antibodies, antibody fragments, antibody mixtures, viral particles, membranes, multicomponent complexes, cells, cell fractions, any fragments thereof, or any combination thereof.

In some embodiments, any of the circular polynucleotides described herein may be prepared according to International patent publication Nos. WO2019118919, WO2020198403, WO2021236952, WO2021236980, WO2021189059, WO2021236855, WO2021226597, and WO2021113777, the entire contents of which are incorporated herein by reference.

In some embodiments, the polynucleotide encodes a tumor antigen polypeptide comprising p53, ART-4, BAGE, ss-catenin /m、Bcr-abL CAMEL、CAP-1、CASP-8、CDC27/m、CDK4/m、CEA、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, MC R, myosin/m, MUC1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 times BCR-abL, plac-1, pml/RARa, PRAME, protease 3, PSA, PSM, RAGE, RU or RU2, SAGE-RT-1/Melan-498, SCG-3, TRP-3, SCG-3, TRP-2, TRP-3, SCP-2, TRP-3, or a combination thereof.

In some embodiments, the polynucleotide encodes an IL2 peptide, IL-2-Ra, anti-CD 19 antibody, anti-CD 20 antibody, chimeric antigen receptor T cell (CAR-T) antibody, anti-HER 2 antibody, etanercept (etanercept) (e.g., enbrel), adalimumab (e.g., semanteb (Humira)), afatin (epoetin alfa) (e.g., epogen), feglastin (filgrastim) (e.g., neupogen), pembrolizumab (e.g., kerdas (Keytruda)), rituximab (rituximab) (e.g., rituximab (Rituxan)), romidepsin (romiplostim) (e.g., nplate), saxaprin (sargramostim) (e.g., leukine), or a variant, fragment, or subunit thereof. Exemplary sequences include those shown in table 3.

An antigenic variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from the wild-type sequence, the native sequence, or a reference sequence. An antigen/polypeptide variant may have substitutions, deletions and/or insertions at certain positions within the amino acid sequence as compared to the native sequence or reference sequence. Typically, the variant has at least 50% identity to the wild-type sequence, the native sequence, or the reference sequence. In some embodiments, the variant has at least 80% or at least 90% identity to a wild-type sequence, a native sequence, or a reference sequence.

The variant antigens/polypeptides encoded by the nucleic acids of the present disclosure may comprise amino acid changes that confer any of a number of desirable properties, e.g., enhancing their immunogenicity, enhancing their expression, and/or improving their stability or PK/PD properties in a subject. Variant antigens/polypeptides may be prepared using conventional mutagenesis techniques and optionally analyzed to determine if they have the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and exemplary such assays are set forth in the examples section. Similarly, PK/PD properties of protein variants may be measured using art-recognized techniques, for example, by determining the expression of antigen in vaccinated subjects over time and/or by observing the persistence of an induced immune response. The stability of the protein encoded by the variant nucleic acid may be measured by determining the thermostability, or stability after urea denaturation, or may be measured using computer prediction. Such experimental and computer-determined methods are known in the art.

The term "identity" refers to the relationship between sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids), as determined by comparing these sequences. Identity also refers to the degree of sequence relatedness between two or more sequences, as determined by the number of matches between segments of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between shorter sequences in two or more sequences, with gaps aligned (if any) being resolved by a specific mathematical model or computer program (e.g., an "algorithm"). Identity of the relevant antigen or nucleic acid can be readily calculated by known methods. "percent identity (%)" when applied to a polypeptide or polynucleotide sequence is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to residues in the amino acid sequence or nucleic acid sequence of the second sequence after aligning the candidate amino acid or nucleic acid sequence with the second sequence and introducing gaps if necessary to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It will be appreciated that identity depends on the calculation of percent identity, but its value may be different due to gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to the particular reference polynucleotide or polypeptide as determined by sequence alignment procedures and parameters described herein and known to those of skill in the art. Such tools for alignment include tools （Stephen F. Altschul, et al (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402）. of the BLAST suite another popular local alignment technique based on the Smith-Waterman algorithm （Smith, T.F.&Waterman, M.S. (1981) "Identification of common molecular subsequences." J. Mol. Biol. 147:195-197）. and a general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm （Needleman, S.B.&Wunsch, C.D. (1970) "A general method applicable to the search for similarities in the amino acid sequences of two proteins." J. Mol. Biol. 48:443-453）., a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) has recently been developed which purportedly produces global alignments of nucleotide and protein sequences faster than other optimal global alignment methods, including Needleman-Wunsch algorithms.

Thus, polynucleotides encoding peptides or polypeptides comprising substitutions, insertions and/or additions, deletions and covalent modifications relative to a reference sequence, in particular a polypeptide (e.g., antigen) sequence disclosed herein, are included within the scope of the present disclosure. For example, a sequence tag or amino acid such as one or more lysines may be added to the peptide sequence (e.g., at the N-terminus or C-terminus). The sequence tags may be used for peptide detection, purification or localization. Lysine can be used to increase peptide solubility or allow biotinylation. Or amino acid residues located in the carboxy and amino terminal regions of the amino acid sequence of the peptide or protein may optionally be deleted, providing a truncated sequence. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, for example, when the sequence is expressed as part of a larger sequence that is soluble or attached to a solid support. In some embodiments, the sequence for (or encoding) a signal sequence, a termination sequence, a transmembrane domain, a linker, a multimerization domain (e.g., a folding region), etc., may be replaced with a replacement sequence that performs the same or a similar function. In some embodiments, the cavities in the protein core may be filled to improve stability, for example, by introducing larger amino acids. In other embodiments, the buried hydrogen bond network may be replaced with a hydrophobic resident to improve stability. In other embodiments, the glycosylation site may be removed and replaced with a suitable residue. Such sequences are readily identifiable by those skilled in the art. It is also to be understood that some of the sequences provided herein comprise sequence tags or terminal peptide sequences (e.g., at the N-or C-terminus), which may be deleted, for example, prior to use in preparing RNA (e.g., mRNA) vaccines.

As will be appreciated by those skilled in the art, protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of coronavirus antigens of interest. For example, provided herein are any protein fragment of a reference protein (representing a polypeptide sequence that is at least one amino acid residue shorter than the reference antigen sequence but otherwise identical) provided that the fragment is immunogenic and confers a protective immune response to coronavirus. In addition to the same but truncated variants as the reference protein, in some embodiments, the antigen comprises 2,3,4, 5, 6, 7, 8, 9, 10 or more mutations, as shown in any of the sequences provided or referenced herein. The antigen/antigen polypeptide may be about 4, 6 or 8 amino acids in length to a full-length protein.

In some embodiments, the polynucleotide is mRNA. Messenger RNA (mRNA) is any RNA that encodes a (at least one) protein (naturally occurring, non-naturally occurring or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. The skilled artisan will appreciate that unless otherwise indicated, the nucleic acid sequences set forth in the present application may be described as "T" in a representative DNA sequence, but where the sequence represents RNA (e.g., mRNA), the "T" will be replaced by "U". Thus, any DNA disclosed and identified herein by a particular sequence identifier also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U".

In some embodiments, the polynucleotide (e.g., mRNA) has an Open Reading Frame (ORF) encoding a cancer antigen. An Open Reading Frame (ORF) is a continuous DNA or RNA segment that begins with an initiation codon (e.g., methionine (ATG or AUG)) and ends with a termination codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). The ORF generally encodes a protein. The sequence may also comprise further elements, such as 5 'and 3' UTRs.

In some embodiments, the RNA (e.g., mRNA) further comprises a 5' UTR, a 3' UTR, a poly (a) tail, and/or a 5' cap analog.

In some embodiments, the mRNA is (a) a DNA molecule, or (b) an RNA molecule. In mRNA, T is optionally replaced by U.

In some embodiments, the mRNA is an RNA molecule. The RNA molecule may also comprise a 5' cap. The 5' Cap may have a Cap 1 structure, a Cap 1 (m 6A) structure, a Cap 2 structure, a Cap 3 structure, a Cap 0 structure, or any combination thereof.

In some embodiments, the polynucleotide is an mRNA encoding an IL-2 molecule. In one embodiment, the IL-2 molecule includes a naturally occurring IL-2 molecule, a fragment of a naturally occurring IL-2 molecule, or a variant thereof. In one embodiment, the IL-2 molecule includes a variant of a naturally occurring IL-2 molecule (e.g., an IL-2 variant, such as described herein) or a fragment thereof.

In one embodiment, the polynucleotide is an mRNA encoding an IL-2 molecule comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of an IL-2 molecule provided in any of tables I-III.

In some embodiments, the mRNA comprises a 3' UTR. In some embodiments, the 3' UTR comprises one or more sequences derived from an amino-terminal split enhancer (AES). In some embodiments, the 3' UTR comprises a sequence derived from a mitochondrially encoded 12S mRNA (mtRNRl).

TABLE V exemplary IL-2 sequences, human Serum Albumin (HSA) sequences and HSA-IL-2 sequences

Table VI exemplary CTLA-4 binding sequences and IL-2 CTLA-4 sequences

Table VII exemplary IL-2 construct sequences

Note that "G5" means that all uracil (U) in mRNA is replaced with N1-methyl pseudouridine.

Stabilization element

Naturally occurring eukaryotic mRNA molecules may contain stabilizing elements, including but not limited to untranslated regions (UTRs) at their 5 'end (5' UTR) and/or their 3 'end (3' UTR), in addition to other structural features such as a 5 'cap structure or a 3' -poly (a) tail. Both the 5 'UTR and the 3' UTR are usually transcribed from genomic DNA and are elements of immature mRNA. During mRNA processing, characteristic structural features of mature mRNA, such as the 5 '-cap and 3' -poly (a) tail, are typically added to transcribed (immature) mRNA.

In some embodiments, the polynucleotide has an open reading frame encoding at least one antigenic polypeptide with at least one modification, at least one 5' end cap, and is formulated within a lipid nanoparticle. During the in vitro transcription reaction, 5 'capping of the polynucleotide may be accomplished simultaneously using chemical RNA cap analogs according to manufacturer's protocol to generate 5 '-guanosine cap structures of 3' -O-Me-m7G (5 ') ppp (5') G [ ARCA cap ]; G (5 ') ppp (5') A; G (5 ') ppp (5') G; m7G (5 ') ppp (5') A; m7G (5 ') ppp (5') G (NEW ENGLAND BioLabs, ipswire, mass.). 5' capping of the modified RNA to produce the "Cap 0" structure m7G (5 ') ppp (5 ') G (NEW ENGLAND BioLabs, ipswich, mass.) can be accomplished post-transcriptionally using vaccinia virus capping Enzyme (VACCINIA VIMS CAPPING Enzyme). Cap 1 structures can be produced using vaccinia virus capping enzyme and 2'-0 methyltransferase to produce m7G (5') ppp (5 ') G-2' -O-methyl. Cap2 structures can be generated using 2' -O methyltransferases to 2' -0 methylate the 5' third last nucleotide on the basis of Cap 1 structures. Cap3 structures can be generated by 2' -O-methylation of the 5' penultimate nucleotide using a 2' -O methyltransferase based on the Cap2 structure. The enzyme may be derived from recombinant sources.

The 3'-poly (A) tail is typically a segment of adenine nucleotide added to the 3' end of transcribed mRNA. In some cases, it may comprise up to about 400 adenine nucleotides. In some embodiments, the length of the 3' -poly (a) tail can be a fundamental element related to the stability of an individual mRNA.

In some embodiments, the polynucleotide comprises a stabilizing element. The stabilizing element may comprise, for example, a histone stem loop. A stem-loop binding protein has been identified (SLBP), which is a 32 kDa protein. It is associated with a histone stem loop at the 3' end of histone messengers in the nucleus and cytoplasm. Its expression level is regulated by the cell cycle and peaks during the S phase, when histone mRNA levels are also elevated. This protein has been shown to be necessary for efficient processing of the 3' end of histone pre-mRNA by U7 snRNP. After processing SLBP remains associated with the stem loop and then stimulates translation of mature histone mRNA in the cytoplasm into group protein proteins. SLBP are conserved in metazoans and protozoans, their binding to the stem loop of histones depends on the loop structure. The minimal binding site comprises at least three nucleotides at 5 'and two nucleotides at 3' relative to the stem loop.

In some embodiments, the polynucleotide (e.g., mRNA) comprises a coding region, at least one histone stem loop, and optionally a poly (a) sequence or polyadenylation signal. The poly (A) sequence or polyadenylation signal should generally enhance the expression level of the encoded protein. In some embodiments, the encoded protein is not a histone protein, a reporter protein (e.g., luciferase, GFP, EGFP, b-galactosidase, EGFP), or a marker or a selectin (e.g., α -globulin, galactokinase, and xanthine: guanine Phosphoribosyl Transferase (GPT)).

In some embodiments, the polynucleotide (e.g., mRNA) comprises a poly (a) sequence or a combination of polyadenylation signals and at least one histone stem loop, although both are essentially representative of alternative mechanisms, the combination thereof synergistically acts to increase protein expression beyond that observed with either element alone. The synergistic effect of the combination of poly (A) and at least one histone stem loop is not dependent on the order of the elements or the length of the poly (A) sequence. In some embodiments, the RNA (e.g., mRNA) does not comprise a Histone Downstream Element (HDE). "histone downstream element" (HDE) comprises a purine-rich polynucleotide segment of about 15 to 20 nucleotides 3' of the naturally occurring stem loop, representing the binding site of U7 snRNA, while U7 snRNA is involved in the processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not comprise an intron.

The polynucleotide (e.g., mRNA) may or may not comprise enhancer and/or promoter sequences, which may or may not be modified, or may not be activated or inactivated. In some embodiments, the histone stem loop is generally derived from a histone gene and comprises intramolecular base pairing of two adjacent partially or fully reverse complementary sequences separated by a spacer, the spacer consisting of a short sequence forming a loop of the structure. Unpaired loop regions are generally unable to base pair with any of the stem-loop elements. This occurs more often in RNA, as it is a key component of many RNA secondary structures, but may also be present in single stranded DNA. The stability of the stem-loop structure generally depends on the length of the mating region, the number of mismatches or bulges, and the base composition. In some embodiments, wobble base pairing (non Watson-Crick base pairing) may occur. In some embodiments, at least one histone stem loop sequence is 15 to 45 nucleotides in length.

In some embodiments, the polynucleotide (e.g., mRNA) has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES, are unstable sequences found in the 3' UTR. AURES can be removed from the RNA composition. Or AURES may remain in the RNA composition.

Signal peptides

In some embodiments, the polynucleotide (e.g., mRNA) has an ORF encoding a signal peptide fused to a coronavirus antigen. The signal peptide comprises 15-60 amino acids from the N-terminus of the protein, and is normally required for transmembrane transport over the secretory pathway, thus generally controlling the entry of most proteins into the secretory pathway in eukaryotes and prokaryotes. In eukaryotes, signal peptides of a new precursor protein (precursor protein) direct ribosomes to the crude Endoplasmic Reticulum (ER) membrane and initiate transport of growing peptide chains across the membrane for processing. ER processing produces mature proteins in which the signal peptide is cleaved from the precursor protein, typically by ER resident signal peptidases of the host cell, or they remain uncleaved and function as membrane anchors. The signal peptide may also facilitate targeting of the protein to the cell membrane. The signal peptide may be 15-60 amino acids in length. For example, the signal peptide may be 15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59 or 60 amino acids in length. In some embodiments, the signal peptide is 20-60、25-60、30-60、35-60、40-60、45-60、50-60、55-60、15-55、20-55、25-55、30-55、35-55、40-55、45-55、50-55、15-50、20-50、25-50、30-50、35-50、40-50、45-50、15-45、20-45、25-45、30-45、35-45、40-45、15-40、20-40、25-40、30-40、35-40、15-35、20-35、25-35、30-35、15-30、20-30、25-30、15-25、20-25 or 15-20 amino acids in length.

Signal peptides from heterologous genes that regulate expression of genes other than coronavirus antigens in nature are known in the art and can be tested for desirable properties and then incorporated into the nucleic acids of the present disclosure. In some embodiments, the signal peptide may comprise a signal peptide described in WO 2021/154763 (the entire contents of which are incorporated herein by reference).

Fusion proteins

In some embodiments, the polynucleotide (e.g., mRNA) encodes an antigen fusion protein. Thus, one or more encoded antigens may include two or more proteins (e.g., proteins and/or protein fragments) linked together. Or a protein fused to a protein antigen does not promote a strong immune response to itself, but rather to a coronavirus antigen. In some embodiments, the antigen fusion proteins retain functional properties from each original protein.

Bracket part

In some embodiments, the polynucleotide (e.g., mRNA) encodes a fusion protein comprising a coronavirus antigen linked to a scaffold moiety. In some embodiments, such scaffold moieties confer desirable properties to antigens encoded by the nucleic acids of the disclosure. For example, the scaffold protein may improve the immunogenicity of an antigen, e.g., by altering the structure of the antigen, altering the uptake and processing of the antigen, and/or causing the antigen to bind to a binding partner.

In some embodiments, the scaffold moiety is a protein that can self-assemble into protein nanoparticles that are highly symmetrical, stable, and structurally ordered, with diameters of 10-150 nm, a very suitable size range for optimal interaction with various cells of the immune system.

In some embodiments, a bacterial protein platform may be used. Non-limiting examples of such self-assembling proteins include ferritin, tetrahydropteridine dioxide, and encapsulation proteins (encapulins).

Ferritin is a protein whose primary function is intracellular iron storage. Ferritin consists of 24 subunits, each consisting of a four alpha helix bundle, which self-assembles in an octahedral symmetrical quaternary structure (Cho k.j. Et al J Mol biol 2009; 390:83-98). Several high resolution structures of ferritin have been established, confirming that helicobacter pylori ferritin consists of 24 identical pathogens, whereas in animals there are ferritin light and heavy chains which can be assembled individually or in different ratios into 24 subunit particles （Granier T. et al. J Biol Inorg Chem. 2003; 8:105-111; Fawson D.M. et al. Nature. 1991; 349:541-544）. ferritin self-assembled into nanoparticles with strong thermal and chemical stability. Thus, ferritin nanoparticles are well suited for carrying and exposing antigens.

Fumarazine synthase (fumazine synthase, FS) is also well suited as a nanoparticle platform for antigen display. FS, which is responsible for the penultimate catalytic step in riboflavin biosynthesis, is a single enzyme （Weber S.E. Flavins and Flavoproteins. Methods and Protocols, Series: Methods in Molecular Biology. 2014）.FS that is 150 amino acids in length and consists of β -sheets and tandem α -helices flanking them, found in a wide variety of organisms including archaea, bacteria, fungi, plants and eubacteria. Many different quaternary ammonium structures of FS have been reported, demonstrating their morphological diversity ranging from homopentamers up to symmetrical assemblies of up to 12 pentamers forming a 150A diameter capsid. FS cages of even more than 100 subunits have been described (Zhang X. Et al J Mol biol. 2006; 362:753-77).

Encapsulated proteins, a novel protein cage nanoparticle isolated from Thermotoga maritima (thermophile Thermotoga maritima), can also be used as a platform for antigen presentation on the surface of self-assembled nanoparticles. The encapsulated protein was assembled from 60 copies of the same 31 kDa monomer, with a thin icosahedral t=1 symmetrical cage structure, with inner and outer diameters of 20 nm and 24 nm, respectively (Sutter m et al, nat Struct Mol biol 2008, 15:939-947). Although the exact function of the encapsulation protein in Thermotoga maritima (T.maritima) has not been clearly understood, its crystal structure has recently been resolved and its function is presumed to be a cellular compartment of the encapsulation protein, such as DyP (dye-decolorized peroxidase) and Flp (ferritin-like protein), which is involved in oxidative stress (Rahmanpour R. Et al FEBS J.2013, 280:2097-2104).

In some embodiments, the polynucleotide encodes a coronavirus antigen (e.g., SARS-CoV-2S protein) fused to a folding domain. The folding domain may be obtained, for example, from phage T4 fibrin (see, for example, tao Y, et al Structure 1997 Jun 15; 5 (6): 789-98).

Linker and cleavable peptide

In some embodiments, a polynucleotide (e.g., mRNA) encodes more than one polypeptide, referred to herein as a fusion protein. In some embodiments, the mRNA further encodes a linker located between at least one or each domain of the fusion protein. The linker may be, for example, a cleavable linker or a protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of an F2A linker, a P2A linker, a T2A linker, an E2A linker, and combinations thereof. This family of self-cleaving peptide linkers, known as 2A peptides, has been described in the art (see, e.g., kim, j.h. et al (2011) PLoS ONE 6:el 8556). In some embodiments, the linker is an F2A linker. In some embodiments, the fusion protein comprises three domains with intervening linkers, with the following structure domain-linker-domain.

Cleavable linkers known in the art may be used in connection with the present disclosure. Exemplary such linkers include F2A linkers, T2A linkers, P2A linkers, E2A linkers (see, e.g., WO2017/127750, the entire contents of which are incorporated herein by reference). The skilled artisan will appreciate that other art-recognized linkers may be suitable for use with the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will also appreciate that other polycistronic constructs (mRNA encoding more than one antigen/polypeptide, respectively, within the same molecule) may be suitable for use as provided herein.

Sequence optimization

In some embodiments, the ORF encoding the antigens of the present disclosure are codon optimized. Codon optimization methods are known in the art. For example, ORFs of any one or more of the sequences provided herein may be codon optimized. In some embodiments, codon optimization may be used to match codon frequencies in the target and host organisms to ensure proper folding, to regulate GC content to increase mRNA stability or reduce secondary structure, to minimize tandem repeat codons or consecutive repeats of bases (run) that may impair gene construction or expression, to tailor transcriptional and translational control regions, to insert or remove protein transport sequences, to remove/add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, to add, remove or reorganize protein domains, to insert or delete restriction enzyme sites, to modify ribosome binding sites and mRNA degradation sites, to adjust translation rates to allow multiple domains of the protein to fold properly, or to reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art-non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In some embodiments, the Open Reading Frame (ORF) sequence is optimized using an optimization algorithm.

In some embodiments, the codon optimized sequence shares less than 95% sequence identity with a naturally occurring or wild type sequence ORF (e.g., a naturally occurring or wild type mRNA sequence encoding an antigen). In some embodiments, the codon optimized sequence shares less than 90% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, the codon optimized sequence shares less than 85% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, the codon optimized sequence shares less than 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an antigen). In some embodiments, the codon optimized sequence shares less than 75% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding an antigen).

In some embodiments, the codon optimized sequence shares 65% to 85% (e.g., about 67% to about 85% or about 67% to about 80%) sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen). In some embodiments, the codon optimized sequence shares 65% to 75% or about 80% sequence identity with a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a coronavirus antigen).

In some embodiments, the antigen encoded by the codon-optimized sequence has the same immunogenicity or a higher immunogenicity (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200%) as compared to the coronavirus antigen encoded by the non-codon-optimized sequence. When transfected into a mammalian host cell, the modified mRNA has stability for 12-18 hours or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours, and is capable of being expressed by the mammalian host cell.

In some embodiments, the codon optimized RNA can be RNA in which G/C levels are increased. The G/C content of a nucleic acid molecule (e.g., mRNA) can affect the stability of RNA. RNA with increased amounts of guanine (G) and/or cytosine (C) residues may be functionally more stable than RNA comprising a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02/098443 discloses a pharmaceutical composition comprising mRNA stabilized by sequence modification in the translation region. Due to the degeneracy of the genetic code, modifications work by replacing existing codons with those which promote higher RNA stability without altering the resulting amino acid. The method is limited to the coding region of RNA.

Non-chemically modified nucleotides

In some embodiments, the polynucleotide (e.g., mRNA) is not chemically modified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of a polynucleotide (e.g., mRNA) include standard nucleoside residues, such as the nucleoside residues (e.g., A, G, C or U) present in the transcribed RNA. In some embodiments, the nucleotides and nucleosides of a polynucleotide (e.g., mRNA) include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT).

Chemical modification

In some embodiments, a polynucleotide (e.g., mRNA) comprises an RNA having an open reading frame encoding a coronavirus antigen, wherein the nucleic acid comprises modified nucleotides and/or nucleosides that may be standard (unmodified) or known in the art. In some embodiments, the nucleotides and nucleosides of a polynucleotide (e.g., mRNA) include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications may include modifications at the sugar, backbone or nucleobase portion of the nucleotide and/or nucleoside as recognized in the art.

The nucleic acid of a polynucleotide (e.g., mRNA) can comprise standard nucleotides and nucleosides, naturally occurring nucleotides and nucleosides, non-naturally occurring nucleotides and nucleosides, or any combination thereof.

In some embodiments, the nucleic acids of the polynucleotides (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) comprise a plurality (more than one) of different types of standard and/or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid comprises one, two, or more (optionally different) types of standard and/or modified nucleotides and nucleosides.

In some embodiments, the modified RNA nucleic acid (e.g., modified mRNA nucleic acid) exhibits reduced degradation in a cell or organism, respectively, after introduction into the cell or organism relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, the modified RNA nucleic acid (e.g., modified mRNA nucleic acid) may exhibit reduced immunogenicity (e.g., reduced innate response) in a cell or organism, respectively, after introduction into the cell or organism relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, the nucleic acid (e.g., RNA nucleic acid, e.g., mRNA nucleic acid) comprises non-natural modified nucleotides that are introduced during or after synthesis of the nucleic acid to achieve a desired function or property. Modifications may be present on internucleotide linkages, purine or pyrimidine bases or sugars. The modification may be introduced at the end of the strand or at any other position in the strand by chemical synthesis or by a polymerase. Any region of the nucleic acid may be chemically modified.

The present disclosure provides modified nucleosides and nucleotides of nucleic acids (e.g., RNA nucleic acids, e.g., mRNA nucleic acids). "nucleoside" refers to a compound comprising a sugar molecule (e.g., pentose or ribose) or derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or derivative thereof (also referred to herein as a "nucleobase"). "nucleotide" refers to a nucleoside comprising a phosphate group. Modified nucleotides may be synthesized by any useful method, such as chemical, enzymatic or recombinant, to include one or more modified or unnatural nucleosides. The nucleic acid may comprise one or more regions of linked nucleosides. Such regions may have variable backbone linkages. The bond may be a standard phosphodiester bond, in which case the nucleic acid will comprise a region of nucleotides.

Modified nucleotide base pairing includes not only standard adenosine-thymine, adenosine-uracil or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of the hydrogen bond donor and hydrogen bond acceptor allows hydrogen bonding to occur between the non-standard base and standard base (e.g., in those nucleic acids having at least one chemical modification) or between two complementary non-standard base structures. An example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any combination of bases/sugars or linkers can be incorporated into the nucleic acids of the disclosure.

In some embodiments, the polynucleotide (e.g., mRNA) comprises uridine at one or more or all uridine positions of the nucleic acid. In some embodiments, mRNA is uniformly modified (e.g., fully modified, modified throughout the sequence) for a particular modification. For example, the nucleic acid may be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, nucleic acids may be uniformly modified for any type of nucleotide residue present in the sequence by substitution with modified residues (e.g., modified residues described above).

The nucleic acid of a polynucleotide (e.g., mRNA) may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., any one or more or all of purine or pyrimidine, or A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure or in a predetermined sequence region thereof (e.g., in an mRNA that includes or does not include a poly (a) tail). In some embodiments, all nucleotides X in a nucleic acid of the disclosure (or in a sequence region thereof) are modified nucleotides, wherein X can be any of nucleotides A, G, U, C, or any of the combinations a+ G, A + U, A + C, G + U, G + C, U + C, A +g+ U, A +g+ C, G +u+c or a+g+c.

The nucleic acid may comprise about 1% to about 100% (relative to the total nucleotide content, or relative to any one or more of the types of nucleotides, A, G, U or C) or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 80%, 95% to 95%, and 95% to 100% nucleotides. It should be understood that any remaining percentages are calculated from unmodified A, G, U or C present.

The mRNA may comprise at least 1% and at most 100% modified nucleotides or any intermediate percentage, for example at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides or at least 90% modified nucleotides. For example, the nucleic acid may comprise a modified pyrimidine, such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracils in the nucleic acid are replaced with modified uracils (e.g., 5-substituted uracils). The modified uracil can be replaced with a compound having a single unique structure, or can be replaced with multiple compounds having different structures (e.g., 2, 3, 4, or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosines in the nucleic acid are replaced with modified cytosines (e.g., 5-substituted cytosines). The modified cytosine may be replaced by a compound having a single unique structure, or may be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4, or more unique structures).

Untranslated region (UTR)

A polynucleotide (e.g., mRNA) may comprise one or more regions or portions that serve or function as untranslated regions. Where the mRNA is designed to encode at least one antigen of interest, the nucleic acid may comprise one or more of these untranslated regions (UTRs). The wild-type untranslated region of a nucleic acid is transcribed but not translated. In mRNA, the 5 'UTR starts from the transcription initiation site and continues to the initiation codon, but does not include the initiation codon, while the 3' UTR starts immediately after the termination codon and continues until the transcription termination signal. There is increasing evidence that UTR plays a regulatory role in nucleic acid molecule stability and translation. Regulatory features of UTRs may be incorporated into polynucleotides of the present disclosure to enhance stability of molecules, etc. Specific features may also be incorporated to ensure controlled down-regulation of transcripts to prevent their misdirection to undesired organ sites. Multiple 5 'UTR and 3' UTR sequences are known and available in the art.

The 5 'UTR is the region of the mRNA immediately upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5' UTR does not encode proteins (is non-coding). The natural 5' UTR has a characteristic of playing a role in translation initiation. They have features such as Kozak sequences, which are well known to be involved in the process of ribosome initiation of many gene translations. The Kozak sequence has the consensus CCR (a/G) CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another 'G'. It is also known that the 5' UTR forms a secondary structure involved in elongation factor binding.

In some embodiments, the 5' UTR is a heterologous UTR, i.e., a UTR associated with a different ORF found in nature. In another embodiment, the 5' UTR is a synthetic UTR, i.e., is not found in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties (e.g., increase gene expression) as well as fully synthetic UTRs. Exemplary 5' UTRs include Xenopus (Xenopus) or human derived a-or b-globulins (8278063; 9012219), human cytochrome b-245 a polypeptides, and hydroxysteroid (17 b) dehydrogenases and tobacco etch virus (Tobacco etch virus) (U.S. 8278063, 9012219, incorporated herein by reference in its entirety). The CMV immediate early 1 (IE 1) gene (US 2014/0206753, WO2013/185069, the entire contents of which are incorporated herein by reference), sequence GGGAUCCUACC (WO 2014/144196) (SEQ ID NO: 89) may also be used. In another embodiment, the 5 'UTR of the TOP gene is the 5' UTR of the TOP gene lacking the 5 'TOP motif (oligopyrimidine stretch) (e.g., WO/2015/101414, W02015/101415, WO/2015/062738, WO 2015/024667), the 5' UTR element derived from the ribosomal protein large 32 (L32) gene (WO/2015/101414, W02015/101415, WO/2015/062738), the 5 'UTR element derived from the 5' UTR of the hydroxysteroid (17-B) dehydrogenase 4 gene (HSD 17B 4) (WO 2015/024667), or the 5 'UTR element derived from the 5' UTR of ATP5A1 (WO 2015/024667) may be used. In some embodiments, an Internal Ribosome Entry Site (IRES) is used in place of the 5' UTR.

The 3 'UTR is the region of mRNA immediately downstream (3') of the stop codon (the codon in the mRNA transcript that signals the termination of translation). The 3' UTR does not encode proteins (is non-coding). It is known that natural or wild-type 3' UTRs have embedded therein segments of adenosine and uridine. These AU-rich features are particularly prevalent in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes (Chen et al, 1995) class I AREs contain several discrete copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA (U/A) (U/A) nonamers. Molecules comprising such AREs include GM-CSF and TNF- α. Class III ARES is less well defined. These U-rich regions do not contain the AUUUA motif. c-Jun and myogenin are two well-studied examples of this class.

Most proteins that bind ARE known to disrupt messenger stability, whereas members of the ELAV family, particularly HuR, have been shown to increase mRNA stability. HuR binds all three classes of ARE. Engineering a HuR specific binding site into the 3' UTR of a nucleic acid molecule will result in HuR binding, thereby stabilizing the in vivo message.

The introduction, removal or modification of 3' UTR-enriched AU elements (ARE) can be used to modulate the stability of a polynucleotide (e.g. mRNA). When engineering a particular nucleic acid, one or more copies of an ARE can be introduced to destabilize the nucleic acids of the disclosure, thereby inhibiting translation and reducing production of the resulting protein. Also, ARE can be identified and removed or mutated to increase intracellular stability, thereby increasing translation and production of the resulting protein. Transfection experiments can be performed in related cell lines using the nucleic acids of the present disclosure, and protein production can be measured at various time points after transfection. For example, cells can be transfected with different ARE engineering molecules and the proteins produced 6 hours, 12 hours, 24 hours, 48 hours and 7 days after transfection ARE determined by ELISA kit using the relevant proteins.

The 3' UTR may be heterologous or synthetic.

One of ordinary skill in the art will appreciate that heterologous or synthetic 5 'UTRs may be used with any desired 3' UTR sequence. For example, a heterologous 5' UTR may be used with a synthetic 3' UTR or a heterologous 3' UTR.

Non-UTR sequences may also be used as regions or sub-regions within a nucleic acid. For example, introns or portions of intronic sequences may be incorporated into regions of nucleic acids of the disclosure. Incorporation of an intron sequence can increase protein production and nucleic acid levels.

Combinations of features may be included in the flanking regions and may be included within other features. For example, an ORF may flank a 5 'UTR, which may contain a strong Kozak translation initiation signal, and/or a 3' UTR, which may include an oligo (dT) sequence for templated addition of a poly-A tail. The 5 'UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes, for example the 5' UTR described in U.S. patent application publication No. 2010/0293625 and PCT/US2014/069155 (the entire contents of which are incorporated herein by reference). It will be appreciated that any UTR from any gene may be incorporated into a region of nucleic acid. In addition, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs that are not variants of the wild-type region. These UTRs, or portions thereof, may be placed in the same orientation as in the transcripts from which they were selected, or the orientation or position may be changed. Thus, a 5 'or 3' UTR may be inverted, shortened, lengthened, made from one or more other 5 'UTRs or 3' UTRs. As used herein, the term "altering" when in relation to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. For example, a 3 'UTR or 5' UTR may be altered relative to a wild-type or natural UTR by a change in orientation or position as taught above, or may be altered by including additional nucleotides, deletions of nucleotides, exchanges of nucleotides, or transposition. Any of these changes that result in an "altered" UTR (whether 3 'or 5') comprise a variant UTR.

In some embodiments, dual, triple, or quad UTRs may be used, such as 5 'UTR or 3' UTR. As used herein, a "dual" UTR is a UTR in which two copies of the same UTR are encoded in tandem or substantially in tandem. For example, the bis- β -globulin 3' UTR may be used as described in U.S. patent publication 2010/0129877 (the contents of which are incorporated herein by reference in their entirety).

It is also within the scope of the present disclosure to have a patterned UTR. As used herein, "patterned UTRs" are those UTRs that reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or abcapcabc, or variants thereof that repeat once, twice, or more than 3 times. In these modes, each letter A, B or C represents a different UTR at the nucleotide level.

In some embodiments, the flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, a polypeptide of interest may belong to a family of proteins expressed in a particular cell, tissue, or at some time during development. UTRs from any of these genes may be exchanged for any other UTRs of the same or different protein family to create new polynucleotides. As used herein, "protein family" is used in its broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, feature, localization, origin, or expression pattern.

The untranslated region may also include a Translation Enhancer Element (TEE). As one non-limiting example, the TEE may include the TEE described in U.S. application No. 2009/0226470 (incorporated herein by reference in its entirety) as well as those known in the art. In vitro transcription of RNA cDNA encoding the polynucleotides described herein can be transcribed using an In Vitro Transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International publication WO 2014/152027, the entire contents of which are incorporated herein by reference. In some embodiments, the RNAs of the present disclosure are prepared according to any one or more of the methods described in WO 2018/053209 and WO 2019/036682 (each of which is incorporated herein by reference).

In some embodiments, the RNA transcript is generated in an in vitro transcription reaction using a non-amplified linear DNA template to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of an RNA polynucleotide (e.g., without limitation, coronavirus mRNA). In some embodiments, the cells, e.g., bacterial cells, e.g., E.coli, e.g., DH-1 cells, are transfected with the plasmid DNA template. In some embodiments, transfected cells are cultured to replicate plasmid DNA, which is then isolated and purified. In some embodiments, the DNA template comprises an RNA polymerase promoter, such as a T7 promoter located 5' of the gene of interest and operably linked to the gene of interest.

In some embodiments, the in vitro transcription template encodes a 5 'untranslated region (UTR), comprises an open reading frame, and encodes a 3' UTR and a poly (a) tail. The specific nucleic acid sequence composition and length of an in vitro transcribed template will depend on the mRNA encoded by the template.

"5 'Untranslated region" (UTR) refers to the region of mRNA immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome), which does not encode a polypeptide. When generating RNA transcripts, the 5' UTR may comprise the promoter sequence. Such promoter sequences are known in the art. It will be appreciated that such promoter sequences will not be present in the compositions of the present disclosure.

"3 'Untranslated region" (UTR) refers to the region of mRNA immediately downstream (i.e., 3') of a stop codon (i.e., a codon in an mRNA transcript that signals the termination of translation) that does not encode a polypeptide.

An "open reading frame" is a continuous DNA segment that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA) and encodes a polypeptide.

A "poly (A) tail" is an mRNA region comprising a plurality of consecutive adenosine monophosphates located downstream, e.g., immediately downstream (i.e., 3 '), of the 3' UTR. The poly (a) tail may comprise 10 to 300 adenosine monophosphates. For example, the poly (A) tail may comprise 10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290 or 300 adenosine monophosphates. In some embodiments, the poly (a) tail comprises 50 to 250 adenosine monophosphates. In a related biological environment (e.g., in a cell, in vivo), the poly (a) tail serves to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and to aid in transcription termination and/or export of mRNA from the nucleus and translation.

In some embodiments, the nucleic acid comprises 200 to 3,000 nucleotides. For example, a nucleic acid may comprise 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

In vitro transcription systems typically comprise a transcription buffer, nucleotide Triphosphates (NTPs), an rnase inhibitor, and a polymerase.

NTP may be manufactured internally, may be selected from suppliers, or may be synthesized as described herein. The NTP may be selected from, but is not limited to, NTPs described herein including natural and non-natural (modified) NTPs.

Any number of RNA polymerases or variants can be used in the methods of the present disclosure. The polymerase may be selected from, but is not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and/or mutant polymerases, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of dnase.

In some embodiments, the RNA transcript is capped by enzymatic capping. In some embodiments, the polynucleotide (e.g., mRNA) comprises a 5' end cap, e.g., 7mG (5 ') ppp (5 ') NlmpNp.

Chemical synthesis

Solid phase chemical synthesis. Polynucleotides (e.g., mRNA) may be made in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated process in which molecules are immobilized on a solid support and synthesized stepwise in a reactant solution. Solid phase synthesis can be used to introduce chemical modifications in the nucleic acid sequence at site-specific locations.

Liquid phase chemical synthesis. The synthesis of polynucleotides (e.g., mRNA) by sequential addition of monomer building blocks (blocks) can be performed in the liquid phase.

A combination of synthesis methods. The synthetic methods discussed above each have their own advantages and limitations. Attempts have been made to combine these approaches to overcome the limitations. Such methods are within the scope of the present disclosure. The combined use of solid or liquid phase chemical synthesis with enzymatic ligation provides an efficient method of producing long-chain nucleic acids that cannot be obtained by chemical synthesis alone.

Ligation of nucleic acid regions or subregions

Assembled nucleic acids by ligase may also be used. DNA or RNA ligases facilitate intermolecular ligation of the 5 'and 3' ends of polynucleotide strands by forming phosphodiester bonds. Nucleic acids, such as chimeric polynucleotides and/or circular nucleic acids, may be prepared by ligation of one or more regions or sub-regions. The DNA fragments may be joined by a ligase catalyzed reaction to produce recombinant DNA having different functions. Two oligodeoxynucleotides, one with a 5 'phosphoryl group and the other with a free 3' hydroxyl group, served as substrates for DNA ligase.

Purification

Purification of nucleic acids described herein may include, but is not limited to, nucleic acid purification (clean-up), quality assurance, and quality control. Purification can be performed by methods known in the art such as, but not limited to AGENCOURT square beads (Beckman Coulter Genomics, danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON Inc, vedbaek, denmark) or HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in connection with a nucleic acid, e.g. "purified nucleic acid", refers to a nucleic acid that is separated from at least one contaminant. A "contaminant" is any substance that renders another substance unsuitable, impure, or inferior. Thus, purified nucleic acids (e.g., DNA and RNA) exist in a form or environment that is different from the form or environment in which they were found in nature, or from the form or environment in which they existed prior to subjecting them to the treatment or purification methods.

Quality assurance and/or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

In some embodiments, the nucleic acid may be sequenced by methods including, but not limited to, reverse transcriptase-PCR.

Quantification of

In some embodiments, polynucleotides (e.g., mRNA) may be quantified in exosomes or when derived from one or more bodily fluids. Body fluids include peripheral blood, serum, plasma, ascites, urine, cerebral Spinal Fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid (cowper's fluid), or periejaculatory fluid, sweat, stool, hair, tears, cyst fluid, pleural and peritoneal fluids, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, fecal water, pancreatic fluids, sinus cavity lavage, bronchopulmonary aspirates, blastocyst (blastocyl) cavity fluid, and cord blood. Or exosomes may be obtained from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver and placenta.

The determination can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, while the exosomes can be isolated using immunohistochemical methods such as enzyme-linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

These methods enable researchers to monitor the level of nucleic acid remaining or delivered in real-time. This is possible because, in some embodiments, the nucleic acids of the present disclosure differ from endogenous forms due to structural or chemical modifications.

In some embodiments, the nucleic acid may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is NANODROP% spectrometer (thermo Fisher, waltham, mass.). The quantified nucleic acid can be analyzed to determine if the nucleic acid is of an appropriate size and to check if the nucleic acid has not degraded. Degradation of nucleic acids may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary Electrophoresis (CE), and Capillary Gel Electrophoresis (CGE).

Therapeutic method

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing and/or treating human and other mammalian cancers. LNMP/RNA (e.g., mRNA or circRNA) formulations can be used as therapeutic or prophylactic agents. They can be used in medicine to prevent and/or treat cancer.

In one embodiment, an LNMP/RNA composition (e.g., an mRNA composition or a circRNA composition) is used to provide prophylactic protection against cancer. Prophylactic protection against cancer can be achieved following administration of an LNMP/RNA composition (e.g., an mRNA or circRNA composition, such as a vaccine). The vaccine may be administered once, twice, three times, four times or more, but it may be sufficient to administer the vaccine once (optionally followed by a single booster). More desirably, the vaccine is administered to an individual suffering from cancer to achieve a therapeutic response. Accordingly, the dosage may need to be adjusted.

In some embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA composition, e.g., vaccine) is administered in a schedule (schedule) of up to two months, up to three months, up to four months, up to five months, up to six months, up to seven months, up to eight months, up to nine months, up to ten months, up to eleven months, up to one year, up to 1.5 years, up to two years, up to three years, or up to four years. The schedules may be the same or different. In some embodiments, the schedule is weekly for the first 3 weeks, then monthly.

The LNMP/RNA composition (e.g., mRNA or circRNA composition, such as a vaccine) can be administered by any route. In some embodiments, the composition is administered by the EVI or IV route.

At any point in the treatment, the patient may be examined to determine if the mutation in the composition is still appropriate. Based on this analysis, the composition may be adjusted or reconfigured to include one or more different mutations or to remove one or more mutations.

Therapeutic and prophylactic compositions

Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing, treating, or diagnosing cancer in humans and other mammals.

In some embodiments, LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions, such as vaccines) can be used to initiate immune effector cells, e.g., to activate Peripheral Blood Mononuclear Cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

In exemplary embodiments, an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotide translated in vivo to produce an antigen polypeptide.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be induced for translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue, or organism. In exemplary embodiments, such translation occurs in vivo, but embodiments are contemplated in which such translation occurs ex vivo, in culture, or in vitro. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of a composition comprising an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) comprising a polynucleotide having at least one translatable region encoding an antigenic polypeptide.

An "effective amount" of an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) is provided based at least in part on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., size and extent of nucleoside modification), and other components of the LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition), as well as other determinants. In general, an effective amount of an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) is one that is more effective than a composition comprising a corresponding unmodified polynucleotide encoding the same antigen or peptide antigen, as the antigen in the cell is produced to provide an induced or enhanced immune response. The increase in antigen production can be demonstrated by an increase in cell transfection (percentage of cells transfected with LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions)), an increase in protein translation of the polynucleotide, a decrease in nucleic acid degradation (e.g., as demonstrated by an increase in the duration of protein translation of the modified polynucleotide), or an altered antigen-specific immune response of the host cell.

In some embodiments, LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be used to treat cancer.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization regimen or during active cancer early in the cancer or after onset of symptoms. In some embodiments, the amount of LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) provided to a cell, tissue, or subject can be an effective immunoprophylaxis amount.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be administered with other prophylactic or therapeutic compounds. As one non-limiting example, the prophylactic or therapeutic compound can be an immunopotentiator, adjuvant or booster. As used herein, when referring to a composition, such as a vaccine, the term "booster" refers to a prophylactic (vaccine) composition that is additionally administered. The booster (or booster vaccine) may be administered after an earlier administration of the prophylactic composition. The time between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

In one embodiment, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) can be administered intramuscularly or intradermally.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be utilized in a variety of environments, depending on the severity of the cancer or the extent or level of unmet medical need. As one non-limiting example, LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be utilized to treat cancer at any stage. LNMP/mRNA or circRNA therapeutic compositions) have excellent properties because they produce greater antibody titers, T cell responses, and earlier onset responses than commercially available anti-cancer vaccines. While not wishing to be bound by theory, LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) have been better designed as mRNA to produce a suitable protein conformation upon translation because LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) utilize natural cellular mechanisms. Unlike traditional vaccines that are manufactured ex vivo and may elicit unwanted cellular responses, LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) are presented to the cellular system in a more natural manner.

A non-limiting list of cancers that can be treated by LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) is set forth below. The peptide epitope or antigen may be derived from any antigen of these cancers or tumors. Such epitopes are known as cancer or tumor antigens. Cancer cells may differentially express cell surface molecules during different stages of tumor progression. For example, cancer cells may express cell surface antigens in benign states, but down-regulate that particular cell surface antigen upon metastasis. Thus, it is contemplated that a tumor or cancer antigen may include an antigen produced during any stage of cancer progression. The method of the present invention can be adapted to accommodate these variations. For example, several different LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be generated for a particular patient. For example, the first vaccine may be used at the beginning of the treatment. At a later point in time, a new LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) can be generated and administered to the patient to treat the different antigens expressed.

Provided herein are pharmaceutical compositions comprising LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions), optionally in combination with one or more pharmaceutically acceptable excipients.

The LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) can be formulated or administered alone or in combination with one or more other components. For example, an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) can comprise other components, including but not limited to immunopotentiators (e.g., adjuvants). In some embodiments, the LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) do not comprise an immunopotentiator or adjuvant (i.e., they do not comprise an immunopotentiator or adjuvant).

In other embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) can be combined with any other therapy useful in treating a patient. For example, a patient can be treated with an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) and an anti-cancer therapeutic agent. Thus, in one embodiment, the methods of the invention may be used in combination with one or more cancer therapeutic agents, e.g., in combination with an anti-cancer therapeutic agent, a traditional cancer vaccine, chemotherapy, radiation therapy, etc. (e.g., simultaneously or as part of an overall therapeutic procedure). The cancer treatment parameters that may be varied include, but are not limited to, dose, timing or duration of administration or therapy, and the dose, timing or duration of cancer treatment may vary. Another treatment for cancer is surgery, which may be used alone or in combination with any existing treatment. Any known agent or therapy (e.g., conventional cancer vaccine, chemotherapy, radiation therapy, surgery, hormonal therapy, and/or biological therapy/immunotherapy) that is useful or has been used or is currently being used to prevent or treat cancer may be used in combination with the compositions according to the invention described herein. One of ordinary skill in the medical arts can determine an appropriate treatment for a subject.

Examples of such agents (i.e., anticancer therapeutic agents) include, but are not limited to, DNA-interacting agents, including, but not limited to alkylating agents (e.g., nitrogen mustards such as chlorambucil (Chlorambucil), cyclophosphamide (Cyclophosphamide), ifosfamide (Isofamide), nitrogen mustards (Mechlorethamine), melphalan (MELPHALAN), uramustine (Uracil mustard), aziridines (Aziridine) such as thiotepa (Thiotepa), mesylate esters such as busulfan (Busulfan), nitrosoureas such as carmustine (Carmustine), Lomustine (Lomustine), streptozocin (Streptozocin), platinum complexes such as Cisplatin (CISPLATIN), carboplatin (Carboplatin), bioreductive alkylating agents such as mitomycin (Mitomycin), procarbazine (Procarbazine), dacarbazine (Dacarbazine) and altretamine (ALTRETAMINE)), DNA strand breaking agents such as bleomycin (Bleomycin), intercalating topoisomerase II inhibitors such as intercalators (Intercalator) such as amsacrine (AMSACRINE), actinomycin D (Dactinomycin), daunorubicin (Daunorubicin), doxorubicin (Doxorubicin), idarubicin (Idarubicin), Mitoxantrone (Mitoxantrone), as well as non-intercalating agents such as etoposide (Etoposide) and teniposide (Teniposide), non-intercalating topoisomerase II inhibitors such as etoposide (Etoposide) and teniposide (Teniposide), and DNA minor groove binders such as Plicamycin (PLICAMYDIN), antimetabolites including but not limited to folic acid antagonists such as Methotrexate (Methotrexate) and trimethamide (trimethoxate), pyrimidine antagonists such as fluorouracil (Fluorouracil), Fluorouridine (Fluorodeoxyuridine), CB3717, azacytidine (Azacitidine) and fluorouridine (Floxuridine), purine antagonists such as mercaptopurine (Mercaptopurine), 6-thioguanine (6-Thioguanine), pravastatin (Pentostatin), sugar modifying analogs such as cytarabine (Cytarabine) and fludarabine (Fludarabine), and ribonucleotide reductase inhibitors such as hydroxyurea, tubulin interactive agents including, but not limited to, colchicine, Vincristine (Vincristine) and vinblastine (Vinblastine), alkaloids and Paclitaxel (Paclitaxel) and cyclophosphamide (Cytoxan), hormonal agents including but not limited to estrogens, conjugated estrogens and ethinyl estradiol (Ethinyl Estradiol) and diethylstilbestrol (Diethylstilbesterol), clocleestrol (Chlortrianisen) and Ai Dengsi terlo (idenetrol), progestins such as hydroxyprogesterone caproate (Hydroxyprogesterone caproate), Medroxyprogesterone (Medroxyprogesterone) and megestrol (Megestrol), and androgens such as testosterone, testosterone propionate, fluoxytestosterone, methyltestosterone, adrenocortical hormones such as prednisone (Prednisone), dexamethasone (Dexamethasone), methylprednisolone (Methylprednisolone) and prednisolone (Prednisolone), luteinizing hormone releasing hormone agents or gonadotropin releasing hormone antagonists such as leuprolide acetate (leuprolide acetate) and goserelin acetate (goserelin acetate), anti-hormone antigens including but not limited to antiestrogens such as Tamoxifen (Tamoxifen), Antiandrogens such as flutamide (Flutamide), and anti-adrenoceptors such as mitotane (Mitotane) and aminoglutethimide (Aminoglutethimide), cytokines including but not limited to IL-1. α.、IL-1 β、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12、IL-13、IL-18、TGF-β、GM-CSF、M-CSF、G-CSF、TNF-a、TNF-β、LAF、TCGF、BCGF、TRF、BAF、BDG、MP、LIF、OSM、TMF、PDGF、IFN-a、IFN-β、IFN-.γ and uteroglobin (Uteroglobin) (U.S. patent No. 5,696,092), anti-angiogenic agents including but not limited to agents that inhibit VEGF (e.g., other neutralizing antibodies), soluble receptor constructs, and, tyrosine kinase inhibitors, antisense strategies, RNA aptamers and ribozymes against VEGF or VEGF receptors, immunotoxins and coagulants, tumor vaccines and antibodies.

Specific examples of anticancer therapeutic agents include, but are not limited to, acitretin (); aclacinomycin (); acodazole hydrochloride (); the pharmaceutical composition comprises aclidinium ()/in-package, zotehead ()/in-package, bezotehead ()/in-package, bezizanol ()/in-package, bezitane (bizelesin), bleomycin sulfate ()/in-package, brequinar sodium (bre) zium, bromopimin ()/in-package, dacarbazine ()/in-package, carbo-on ()/in-package, carbo-package, valin ()/in-package, bezocine ()/in-package, and/or-package The pharmaceutical composition comprises (a) a compound selected from the group consisting of (a) cinimazamide mesylate, (b) cytarabine, (c) dacarbazine, (d) dacarbazine, (c) dacarbazine hydrochloride, (d) dacarbazine, (c) dexomaplatin, (d) dezaguanosine, (d) fluzaguanosine mesylate, (c) danuquinone, (d) docetaxel, (d) doxorubicin hydrochloride, (c) doxorubicin hydrochloride, (d) droloxifene, (c) droloxifene, (d) droloxifene, (c) droxol hydrochloride, (d) drotazornine, (c) eldronithine hydrochloride, (d) eldrozoprine, (c) eldroplatanum, (c) envone, eldroplatanum (c) eldroplatanum, eldropladine (c) eldroplidine hydrochloride, (d) eldroplicin hydrochloride, (c) eldroglicladine hydrochloride, (d) eldroplicin hydrochloride, (d) fluvoglicladine, eldroplidine (c) etodol) fluvone, eldroplide (c) fludroplide). Fluocitabine (flurocitabine), phosphinoquinol (fosquidone), fos Qu Xingna (fostriecin sodium), gemcitabine (gemcitabine), gemcitabine hydrochloride (gemcitabine hydrochloride), hydroxyurea (hydroxyurea), idarubicin hydrochloride (idarubicin hydrochloride), ifosfamide (ifosfamide), rimoformine (ilmofosine), interleukin II (including recombinant interleukin II or rIL 2), and pharmaceutical compositions containing the same, Interferon alpha-2 a; interferon alpha-2 b; interferon alpha-nl, interferon alpha-n 3, interferon beta-I a, interferon gamma-I b, iproplatin (iproplatin), irinotecan hydrochloride (irinotecan hydrochloride), lanreotide acetate (lanreotide acetate), letrozole (letrozole), leuprorelin acetate (leuprolide acetate), liazome hydrochloride (liarozole hydrochloride), lomef Qu Suona (lometrexol sodium), lomustine (lomustine), ruxoanthraquinone hydrochloride (losoxantrone hydrochloride), maxorolol (masoprocol), maytansine (maytansine), nitrogen mustard hydrochloride (mechlorethamine hydrochloride), megestrol acetate (megestrol acetate), melengestrol acetate (melengestrol acetate), melphalan (melphalan), minoril (menogaril), mercaptopurine (mercaptopurine), methotrexate (methotrexate), methotrexate sodium (methotrexate sodium), metoprolin (metoprine), mezome (meturedepa), mi Ting polyamine (mitindomide), mitomycin (mitocarcin), mi Tuoke (mitocromin), tolin (mitocromin), 2), anthraquinone (mitocromin), and mitomycin (mitocromin), and other mitomycin (mitocromin, and other than the other materials (mitocromin, and the other materials (mitocromin, 393-carrier, mitocromin, and the other materials ) Pelargomycin (); pentamustine (); pelomycin sulfate (); the pharmaceutical composition comprises nipaginamide (); pipobromine (); piposulfan (); piscine hydrochloride (); plicamycin (plicamycin), praziram (); sodium porfilim (sodium sovidium), pofilimycin (porfiromycin); prednisotine (); procarbazine hydrochloride (); puromycin hydrochloride (); flufurlin (); libopurine (); polyglutamine hydrochloride (); plitebufalin (); plitebuxine hydrochloride (); semuslimine hydrochloride (); sodium pap-phosphate (); sepamycin hydrochloride (); spirogermanium hydrochloride (); spiroplakoplatin (spiplatin), streptozocin (); sulchlorurea (); teicoplanin sodium (tectavalan) fluvalin (); thiotefavone (); thiotebufavone (); thiotebufavoglide ()); plifoglide hydrochloride ()) tiazofurin), tirapazamine (tirapazamine), toremifene citrate (toremifene citrate), acetic acid Qu Situo dragon (trestolone acetate), troxiribine phosphate (triciribine phosphate), trimetrazine (trimetrexate), trimetrazine glucuronate (trimetrexate glucuronate), triptorelin (triptorelin), tobrazile hydrochloride (tubulozole hydrochloride), uramestin (uracil mustard), uratepa (uredepa), vatupe (vapreotide), verteporfin (verteporfin), vinblastine sulfate (vinblastine sulfate), vincristine sulfate (VINCRISTINE SULFATE). Vindesine (vindesine), vindesine sulfate (VINDESINE SULFATE), vinepidine sulfate (VINEPIDINE SULFATE), vinglycinate sulfate (VINGLYCINATE SULFATE), vinrosine sulfate (vinleurosine sulfate), vinorelbine tartrate (vinorelbine tartrate), vinrosidine sulfate (vinrosidine sulfate), vinzolidine sulfate (vinzolidine sulfate), vorozole, cinipline (zeniplatin), cilostatin (zinostatin), and zorubicin hydrochloride (zorubicin hydrochloride).

Other anticancer drugs include, but are not limited to, 20-epi-l, 25-dihydroxyvitamin D3, 5-ethynyluracil, angiogenesis inhibitors, anti-backfeed morphogenic protein-1, ara-CDP-DL-PTBA, BCR/ABL antagonists, caRest M, CARN, casein kinase Inhibitors (ICOS), clotrimazole (clotrimazole), colestomycin A (collismycin A), colestomycin B (collismycin B), combretastatin A4 (combretatin A4), cobicidin 816 (crambescidin 816), candididin 8 (cryptosporidium 8), kulasiocin A, dehydroascin B (dehydrodidemnin B), ascin B (didemnin B), dihydro-5-azacytidine (dihydro-5-azacytidine), dihydropaclitaxel (dihydrotaxol), and pharmaceutical compositions containing them, Multi-calico SA (duocarmycin SA), carboxylic Acid (CA) Ha Latai F (kahalalide F), lamellarin-N triacetate (lamellarin-N TRIACETATE), leuprorelin + estrogen + progestin (leuproolide + estrogen + progesterone), lixogram Lin Xianan (lissoclinamide 7), monophosphoryl lipid A + mycobacterial cell wall SK (monophosphoryl lipid A + myobacterium CELL WALL SK), N-acetyldinaline (N-ACETYLDINALINE), N-substituted benzamide, 06-benzyl guanine, prasugrel A (placetin A), prasugrel B (placetin B), platinum complexes, platinum-triamine complexes, re 186 etidronate (rhenium Re etidronate), RII retinoic acid amide (etidronate), gibberelin B1 (etidronate), etidronate, sarcoidol 2, sanguatin (etidronate), age derivative inhibitor 1 (etidronate inland 1), silk mycin (N-ACETYLDINALINE), 5-fluoropyrimidine (F) 5-benzoguanamine), oxaprozin B (etidronate), oxaprozin 2 (etidronate), oxaprozin A (etidronate), oxaprozin B (etidronate), oxaprozidine (etidronate), oxaprozin B (etidronate), and oxaprozin-etidronate (zanoterone) Benincaplatin (zeniplatin) and benzylidene (zilascorb).

The invention also includes administering a composition comprising an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) in combination with radiation therapy, which includes the use of X-rays, gamma rays, and other sources of radiation to destroy cancer cells. In some embodiments, radiation therapy is administered with external beam radiation or remote radiation therapy (where the radiation is from a remote source). In other embodiments, radiation therapy is administered as an internal therapy or brachytherapy (where the radiation source is placed in the body close to the cancer cells or tumor mass).

In particular embodiments, an appropriate anti-cancer regimen is selected according to the type of cancer. For example, a prophylactically or therapeutically effective amount of a composition comprising an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) may be administered to a patient suffering from ovarian cancer in combination with a prophylactically or therapeutically effective amount of one or more other agents useful in therapy of ovarian cancer, including, but not limited to, intraperitoneal radiation therapy such as P32 therapy, whole-abdominal and pelvic radiation therapy, cisplatin, paclitaxel (Taxol) or docetaxel (Taxotere) in combination with cisplatin or carboplatin, cyclophosphamide in combination with cisplatin, cyclophosphamide in combination with carboplatin, 5-FU in combination with folinic acid (leucovorin), etoposide, liposomal Doxorubicin (Doxorubicin), gemcitabine, or topotecan. Cancer therapies and their dosages, routes of administration, and recommended uses are known in the art and have been described in literature such as Physics' S DESK REFERENCE (56 th ed., 2002).

In some embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) is administered with a T cell activator, such as an immune checkpoint modulator. Immune checkpoint modulators include stimulatory checkpoint molecules and inhibitory checkpoint molecules, i.e. anti-CTLA 4 and anti-PDl antibodies. Stimulation checkpoint inhibitors act by facilitating the checkpoint process. Some of the stimulus checkpoint molecules are tumor necrosis factor (T F) receptor superfamily members-CD 27, CD40, OX40, GITR, and CD 137, while others belong to the B7-CD28 superfamily-CD 28 and ICOS. OX40 (CD 134) is involved in the expansion of effector T cells and memory T cells. anti-OX 40 monoclonal antibodies have been shown to be effective in treating advanced cancers. MEDI0562 is a humanized OX40 agonist. GITR is a glucocorticoid-induced T FR family-related gene involved in T cell expansion. Some antibodies to GITR have been shown to promote anti-tumor responses. ICOS (inducible T cell costimulator) is important in T cell effector function. CD27 supports antigen-specific expansion of naive T cells and is involved in T and B cell memory production. Some agonistic anti-CD 27 antibodies are under development. CD122 is the interleukin 2 receptor beta subunit. KTR-214 is a CD 122-biased immunostimulatory cytokine.

Checkpoint inhibiting molecules include, but are not limited to, PD-1, TEVI-3, VISTA, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR and LAG3、PDLl、PDL2、PDl、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD160、CGEN-15049、CHK1、CHK2、A2aR、B-7 family ligands or combinations thereof. Ligands for checkpoint proteins include, but are not limited to ：CTLA-4、PDLl、PDL2、PDl、B7-H3、B7-H4、BTLA、HVEM、TIM3、GAL9、LAG3、VISTA、KIR、2B4、CD 160、CGEN-15049、CHK1、CHK2、A2aR and B-7 family ligands. In some embodiments, the anti-PD-1 antibody is BMS-936558 (nano Wu Liyou mab). In other embodiments, the anti-CTLA-4 antibody is ipilimumab (Iplilimumab) (trade name Yervoy, previously referred to as MDX-010 and MDX-101).

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be combined with anti-cancer therapeutic agents to further enhance the immunotherapeutic response. The LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) and the other therapeutic agent can be administered simultaneously or sequentially. When other therapeutic agents are administered simultaneously, they may be administered in the same formulation or in separate formulations, but at the same time. When the administration of the other therapeutic agent and the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) are spaced apart in time, the other therapeutic agent is administered sequentially to each other and to the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition). The time interval for administration of these compounds may be several minutes, or it may be longer, for example hours, days, weeks, months. For example, in some embodiments, the compounds are administered at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours, or more intervals. In some embodiments, the compounds are administered at 2, 3,4, 5,6, or 7 or more day intervals. In some embodiments, the LNMP/RNA composition (e.g., mRNA therapeutic composition) is administered prior to the anticancer therapeutic. In some embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) is administered after the anticancer therapeutic.

Other therapeutic agents include, but are not limited to, anti-cancer therapeutic agents, adjuvants, cytokines, antibodies, antigens, and the like.

Therapeutic agents may also include therapeutic agents that may treat and/or prevent chronic pain and/or symptoms of chronic pain, including, but not limited to:

(i) Opioid analgesics such as morphine (morphine), heroin (heroin), hydromorphone (hydromorphone), oxymorphone (oxymorphone), levorphanol (levorphanol), levorphanol (levallorphan), methadone (methadone), meperidine (meperidine), fentanyl (fentanyl), cocaine (cocaine), codeine (codeine), dihydrocodeine (dihydrocodeine), oxycodone (oxycodone), hydrocodone (hydrocodone), dextropropoxide (propoxyphene), nalmefene (nalmefene), allylmorphine (nalorphine), naloxone (naloxone), naltrexone (naltrexone), buprenorphine (Buprenorphine), buprenorphine (butorphanol), nalbuphine (nalbuphine) or pentazocine (pentazocine),

(Ii) Non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin (aspirin), diclofenac (dichlofenac), diflunisal (diflusinal), etodolac (etodolac), fenbufen (fenbufen), fenoprofen (fenoprofen), flubensal (flufenisal), flurbiprofen (flurbiprofen), ibuprofen (ibuprofen), indomethacin (indomethacin), ketoprofen (ketoprofen), ketorolac (ketorolac), meclofenamic acid (meclofenamic acid), mefenamic acid (MEFENAMIC ACID), nabumetone (Nabumetone), naproxen (naproxen), oxaprozin (oxaprozin), phenylbutazone (phenylbutazone), piroxicam (piroxicam), sulindac (sulindac), tolmetin (tolmetin) or zomepirac (zomepyrac), mobil (mobic) (meloxicam SR (Meloxicam SR)), or pharmaceutically acceptable salts thereof,

(Iii) Barbital analgesics such as ipratropium (amobarbital), aprazapirne (aprobarbital), butobutyraltital (butabarbital), butobutyraltital (butalbital), tolbital (methobarbital), methylprednisoldine (metalbital), methoprenatal (methohexital), pentobarbital (pentobarbital), phenobarbital (phenobarbital), stavobarbital (secobarbital), tobutobutyraltital (talbutal), thiobarbital (thiamaryl) or thiobarbital (thiopental), or pharmaceutically acceptable salts thereof,

(Iv) Benzodiazepines with analgesic activityA class (benzodiazepine), such as cloazepine (chlordiazepoxide), clorac (chlorazepic acid), diazepam (diazepam), fluoazepam (flurazepam), lorazepam (lorazepam), oxazepam (oxazepam), temazepam (temazepam) or triazolam (triazolam), or a pharmaceutically acceptable salt thereof;

(v) An H1 antagonist having analgesic activity, such as diphenhydramine (DIPHENHYDRAMINE), pyrilamine (pyrilamine), promethazine (promethazine), chlorpheniramine (chlorpheniramine) or chlorocyclidine (chlorcyclidine), or a pharmaceutically acceptable salt thereof;

(vi) Analgesics, such as glimmer (glutethimide), methamphetamine (meprobamate), methodol (methacalone), or ketanserin (dichloralphenazone), or a pharmaceutically acceptable salt thereof;

(vii) Skeletal muscle relaxants such as baclofen (baclofen), carisoprodol (carisoprodol), cloxazone (chlorzoxazone), cyclobenzaprine (cyclobenzaprine), methocarbamol (methocarbamol) or orfrenazine, or a pharmaceutically acceptable salt thereof;

(viii) NMDA receptor antagonists, such as dextromethorphan (dextromethorphan) (+) -3-hydroxy-N-methyl morphinane) or its metabolite dextrorphan (dextrorphan) (+) -3-hydroxy-N-methyl morphinane), ketamine (Ketamine), memantine (memantine), pyrroloquinoline quinone, or cis-4- (phosphonomethyl) -2-piperidinecarboxylic acid, or a pharmaceutically acceptable salt thereof;

(ix) Alpha-adrenergic agents, such as doxazosin, tamsulosin, clonidine or 4-amino-6, 7-dimethoxy-2- (5-methanesulfonamido-1, 2,3, 4-tetrahydroisoquinolin-2-) yl) -5- (2-pyridyl) quinazoline,

(X) Tricyclic antidepressants, such as desipramine (desipramine), promethazine (imipramine), amitriptyline (AMITRIPTYLINE) or nortriptyline (nortriptyline),

(Xi) Anticonvulsants such as carbamazepine (carbamazepine) or valproate (valproate),

(Xii) Tachykinin (NK) antagonists, in particular NK-3, NK-2 or NK-1 antagonists, such as (. Alpha.R, 9R) -7- [3, 5-bis (trifluoromethyl) benzyl ] -8,9,10, 11-tetrahydro-9-methyl-5- (4-methylphenyl) -7H- [1,4] diaza [2,1-g ] [1,7] naphthyridine-6-13-dione (TAK-637), 5- [ [ [ (2R, 3S) -2- [ (1R) -1- [3, 5-bis (trifluoromethyl) phenyl ] ethoxy-3- (4-fluorophenyl) -4-) morpholinyl ] methyl ] -1, 2-dihydro-3H-1, 2, 4-triazol-3-one (MK-869), lanetane (ranepitant), dapitant (dapitant) or (2S, 3S) 3- [ [ 2-methoxy-5- (trifluoromethoxy) phenyl ] methylamino ] -2-phenyl-piperidine,

(Xiii) Muscarinic antagonists such as oxybutynin (oxybutin), tolterodine (tolterodine), propiverine (propiverine), tosipratropium chloride (trospium chloride) or darifenacin (darifenacin),

(Xiv) A COX-2 inhibitor which is a compound selected from the group consisting of, such as celecoxib (celecoxib) rofecoxib or rofecoxib valdecoxib,

(Xv) Non-selective COX inhibitors, preferably inhibitors with GI protection, such as nitroflurbiprofen (nitroflurbiprofen) (HCT-1026),

(Xvi) Coal tar analgesics (coral TAR ANALGESIC), in particular acetaminophen (paracetamol),

(Xvii) Neuroleptics, such as haloperidol (droperidol),

(Xviii) Vanilloid receptor agonists (e.g. resiniferatoxin) or antagonists (e.g. capsazepine),

(Xix) Beta adrenergic agents, such as propranolol (propranolol),

(Xx) Local anesthetics, such as mexiletine (mexiletine),

(Xxi) Corticosteroids, such as dexamethasone (dexamethasone),

(Xxii) 5-hydroxytryptamine (serotonin) receptor agonists or antagonists,

(Xxiii) Cholinergic (nicotinic) analgesics,

(Xxiv) Tramadol TM (TramadolTM) is used as a starting material,

(Xxv) PDEV inhibitors such as sildenafil (sildenafil), vardenafil (vardenafil) or tadalafil (tadalafil).

In some embodiments, provided methods comprise administering an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) in combination with an immune checkpoint modulator. In some embodiments, the immune checkpoint modulator, e.g., checkpoint inhibitor, e.g., anti-PD-1 antibody, is administered at a dosage level sufficient to deliver 100-300 mg to the subject. In some embodiments, the immune checkpoint modulator, e.g., checkpoint inhibitor, e.g., anti-PD-1 antibody, is administered at a dosage level sufficient to deliver 200 mg to the subject. In some embodiments, the immune checkpoint modulator, e.g., checkpoint inhibitor, e.g., anti-PD-1 antibody, is administered by intravenous infusion. In some embodiments, the immune checkpoint modulator is administered to the subject two, three, four or more times. In some embodiments, the immune checkpoint modulator is administered to the subject on the same day that the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) is administered.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be formulated or administered in combination with one or more pharmaceutically acceptable excipients. In some embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) comprises at least one additional active, e.g., a therapeutically active, a prophylactically active, or a combination of both. The LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) can be sterile, pyrogen-free, or sterile and pyrogen-free. General considerations for formulating and/or manufacturing pharmaceutical agents, such as vaccine compositions, can be found, for example, in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21st ed., lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, the LNMP/RNA composition (e.g., mRNA or circRNA therapeutic composition) is administered to a human, human patient, or subject. The phrase "active ingredient" generally refers to an LNMP/RNA composition (e.g., an mRNA or circRNA therapeutic composition) or a polynucleotide contained therein, such as an RNA polynucleotide encoding an antigen polypeptide (e.g., an mRNA polynucleotide or a circRNA polyribonucleotide).

The formulation of the LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) described herein can be prepared by any method known in the pharmacological arts or later developed. Typically, such preparation methods include the steps of associating the active ingredient (e.g., an mRNA polynucleotide or a circRNA polyribonucleotide) with an excipient and/or one or more other adjunct ingredients, and then, if necessary and/or desired, partitioning, shaping and/or packaging the product into the desired single-or multi-dose units.

LNMP/RNA compositions (e.g., mRNA or circRNA therapeutic compositions) can be formulated with one or more excipients to (1) increase stability, (2) increase cell transfection, (3) allow sustained or delayed release (e.g., from depot formulations), (4) alter biodistribution (e.g., targeting a particular tissue or cell type), (5) increase translation of the encoded protein in vivo, and/or (6) alter the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients such as any and all solvents, dispersion media, diluents or other liquid carriers, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, excipients may also include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipid complexes, core shell nanoparticles, peptides, proteins, cells transfected with cancer RNA vaccines (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

Kit for detecting a substance in a sample

The invention also provides a kit comprising a container having an RNA composition (e.g., an mRNA or circRNA therapeutic composition) described herein. The kit may also include instructional materials for applying or delivering the mRNA or circRNA therapeutic composition to a subject according to the methods of the invention. The skilled artisan will appreciate that the instructions for using the RNA composition (e.g., mRNA or circRNA therapeutic composition) in the methods of the invention can be any form of instructions. Such instructions include, but are not limited to, written instruction material (e.g., labels, brochures, leaflets), spoken instruction material (e.g., audiotape or CD), or video instruction (e.g., videotape or DVD). The therapeutic composition (e.g., mRNA or circRNA therapeutic composition) may include material for a single administration (e.g., in a single dose form), or may include material for multiple administrations (e.g., a "multi-dose" kit).

The informative material of the kit is not limited in its form. In one embodiment, the informational material may include information regarding the production of a therapeutic composition (e.g., an mRNA or circRNA therapeutic composition), a drug substance, or a drug product, molecular weight of a composition, a drug substance, or a drug product, concentration, expiration date, batch or production site information, etc., as described herein. In one embodiment, the informational material relates to a method for administering a dosage form of an mRNA or circRNA therapeutic composition. In one embodiment, the informational material relates to a method for administering a dosage form of a circRNA therapeutic composition.

In addition to the dosage form of the mRNA or circRNA therapeutic compositions described herein, the kit may also include other ingredients, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter antagonists or sweeteners), fragrances, dyes or colorants (e.g., to stain or color one or more components of the kit), or other cosmetic grade ingredients, and/or a second agent for treating a condition or disorder described herein. Or the other ingredients may be contained in a kit, but in a composition or container that is different from the mRNA or circRNA therapeutic composition described herein. In such embodiments, the kit can include instructions for mixing a therapeutic composition (e.g., an mRNA or a circRNA therapeutic composition) or a nucleic acid molecule (e.g., an mRNA or a circRNA) described herein with other ingredients, or instructions for using a therapeutic composition (e.g., an mRNA or a circRNA therapeutic composition) or a nucleic acid molecule (e.g., an mRNA or a circRNA) described herein with other ingredients.

In some embodiments, the components of the kit are stored under inert conditions (e.g., under nitrogen or other inert gas such as argon). In some embodiments, the components of the kit are stored under anhydrous conditions (e.g., with a desiccant). In some embodiments, the components are stored in a light shielding container, such as an amber vial.

The dosage forms of the therapeutic compositions (e.g., mRNA or circRNA therapeutic compositions) or nucleic acid molecules (e.g., mRNA or circRNA) described herein may be provided in any form (e.g., liquid, dried, or lyophilized form). Preferably, the therapeutic compositions (e.g., mRNA or circRNA therapeutic compositions) or nucleic acid molecules (e.g., mRNA or circRNA) described herein are substantially pure and/or sterile. When the therapeutic composition (e.g., mRNA or circRNA therapeutic composition) or nucleic acid molecule (e.g., mRNA or circRNA) described herein is provided in a liquid solution, the liquid solution is preferably an aqueous solution, preferably a sterile aqueous solution. When the therapeutic compositions (e.g., mRNA or circRNA therapeutic compositions) or nucleic acid molecules (e.g., mRNA or circRNA) described herein are provided in dry form, reconstitution is typically by addition of a suitable solvent. Solvents such as sterile water or buffers may optionally be provided in the kit.

The kit may include one or more containers for containing the therapeutic compositions of the dosage forms described herein. In some embodiments, the kit comprises separate containers, dividers, or compartments for the composition and the informational material. For example, the therapeutic composition or nucleic acid molecule may be contained in a bottle, vial or syringe, and the informational material may be contained in a plastic sleeve or package. In other embodiments, the individual elements of the kit are contained in a single undivided container. For example, the dosage forms of the therapeutic compositions (e.g., mRNA or circRNA therapeutic compositions) or nucleic acid molecules (e.g., mRNA or circRNA) described herein are contained in a bottle, vial, or syringe to which the informational material is affixed in a labeled form. In some embodiments, the kit comprises a plurality (e.g., a pack) of individual containers, each container comprising one or more unit dosage forms of a therapeutic composition (e.g., an mRNA or a circRNA therapeutic composition) or a nucleic acid molecule (e.g., an mRNA or a circRNA) described herein. For example, the kit comprises a plurality of syringes, ampules, foil packages, or blister packs, each containing a single unit dose of the dosage forms described herein.

The container of the kit may be closed, waterproof (e.g., impermeable to moisture changes or evaporation), and/or opaque.

The kit optionally comprises a device suitable for use of the dosage form, such as a syringe, pipette, forceps, measuring spoon, swab (e.g., a cotton or wood swab) or any such device.

Kits of the invention can include dosage forms of different specifications to provide a subject with a dosage suitable for one or more of the initiation phase regimen, induction phase regimen, or maintenance phase regimen described herein. Or the kit may include scored tablets to allow the user to administer the divided doses as desired.

Examples

The invention will now be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.

EXAMPLE 1 preparation and modification of LNMP and formulation of LNMP containing mRNA

This example describes the preparation of a structural component comprising a natural lipid (e.g., a lipid extracted from a natural source), and modification of the structural component to form a lipid reconstituted natural messenger package (LNMP). One example of an LNMP is LPMP (reconstituted plant messenger package). Preparation of Natural Messenger Package (NMP), modification of NMP to prepare lipid reconstituted natural messenger package (LNMP), and formulation of NMP and LNMP containing mRNA are all illustrated by preparation of PMP and preparation and formulation LPMP, which can be accomplished using the methods disclosed in International patent application publication No. WO 2021/04301, the entire contents of which are incorporated herein by reference.

Specifically, all of the protocols disclosed in examples 1-17 of International patent application publication No. WO 2021/04301 are incorporated herein by reference in their entirety, including example 1. Isolation of plant messenger bags from plants; example 2. Production of purified Plant Messenger Package (PMP), example 3. Characterization of plant messenger package, example 4. Characterization of plant messenger package stability, example 5. Loading PMP with cargo, example 6. Increasing PMP cellular uptake by formulating PMP with ionic liquid, example 7. Modifying PMP with ionizable lipid, example 8. Formulating LPMP with microfluidic technology, example 9. Loading and delivering mRNA into lipid reconstituted PMP with ionizable lipid, example 10. Cellular uptake of native and reconstituted PMP (with or without ionizable lipid modification), example 11. Increasing PMP cellular uptake by formulating PMP with cationic lipid, example 12. Modifying PMP with cationic lipid, example 13. Loading and delivering mRNA into lipid reconstituted PMP with cationic lipid, example 14. Cellular uptake of native and reconstituted PMP (with or without cationic lipid modification), example 15. Loading ratio using cationic lipid 67 and ethyl lipid 67, example 16, and example 17 are optimized for loading ratio of mRNA.

EXAMPLE 2 preparation of mRNA therapeutic composition

Polynucleotide production and characterization

The production of polynucleotides and/or portions or regions thereof may be accomplished using methods taught in international patent application publication No. WO 2014/152027, the entire contents of which are incorporated herein by reference. Purification methods may include methods taught in international patent application publications WO2014/152030 and WO2014/152031 (the entire contents of which are incorporated herein by reference). Methods of detection and characterization of polynucleotides may be performed according to the methods taught in International patent application publication No. WO2014/144039, the entire contents of which are incorporated herein by reference. Characterization of the polynucleotide may be accomplished using polynucleotide alignment, reverse transcriptase sequencing, charge distribution analysis, detection of RNA impurities, or any combination of two or more of the foregoing. For example, "characterizing" includes determining the sequence of an RNA transcript, determining the purity of an RNA transcript, or determining the charge heterogeneity of an RNA transcript. Such methods are taught, for example, in international patent application publications WO2014/144711 and WO2014/144767 (the entire contents of which are incorporated herein by reference).

Additional details of mRNA design and modification can be found in WO 2021/154763 and WO 2021/188969 (the entire contents of which are incorporated herein by reference).

Preparation of mRNA-containing LPMP

General protocol and Experimental design

Protocols and detailed experimental procedures for the preparation of Natural Messenger Packages (NMP), the modification of NMP to prepare lipid reconstituted natural messenger packages (LNMP), and the formulation of mRNA-containing NMP and LNMP have been discussed in example 1, which lists all of the experimental protocols disclosed in examples 1-17 of International patent application publication No. WO 2021/04301 (the entire contents of all examples are incorporated herein by reference). In addition, protocols and detailed experimental steps for the preparation and formulation of LNMP (with or without mRNA) are discussed in international application No. PCT/US22/50571, the entire contents of which are incorporated herein by reference, wherein the natural lipids in LNMP are extracted from bacterial sources. In this example, the protocols and detailed experimental steps were followed in preparing NMP (e.g., with bacterial lipids), LNMP (e.g., with bacterial lipids), PMP, LPMP, LNP, and formulating NMP containing mRNA (e.g., with bacterial lipids), LNMP (e.g., with bacterial lipids), PMP, LPMP, or LNP.

LPMP is used as an example of an LNMP in this embodiment. Briefly, the isolation and purification of crude Plant Messenger Packages (PMPs) from lemon and characterization of these PMPs was performed according to the experimental design and protocols for plant sources described in International patent application publication No. WO 2021/04301, the entire contents of which are incorporated herein by reference, example 1, isolation of plant messenger packages from plants, example 2, production of purified Plant Messenger Packages (PMPs), example 3, plant messenger package characterization, and example 4, characterization of plant messenger package stability.

Modification of native PMP with cholesterol and PEG-lipid or reconstitution of lemon or algae LPMP was performed according to the experimental design and protocols for plant sources described in international patent application publication No. WO 2021/04301 (the entire contents of which are incorporated herein by reference), example 6, increasing PMP cellular uptake by formulating PMP with ionic liquids, example 7, modifying PMP with ionizable lipids, example 10, cellular uptake of native and reconstituted PMP (with or without ionizable lipid modification), example 11, increasing PMP cellular uptake by formulating PMP with cationic lipids, example 12, modifying PMP with cationic lipids, and example 14, cellular uptake of native and reconstituted PMP (with or without cationic lipid modification).

Formulation of PMP with mRNA and lemon lipid or algal lipid reconstitution LPMP was performed according to the experimental design and protocol for nucleic acid loading described in international patent application publication No. WO 2021/04301 (the entire contents of which are incorporated herein by reference), example 5, loading PMP with cargo, example 8, formulation LPMP with microfluidic technology, example 9, loading and delivery of mRNA into lipid reconstituted PMP using ionizable lipids, example 13, loading and delivery of mRNA into lipid reconstituted PMP using cationic lipids, example 15, improved loading using cationic lipids GL67 and ethyl PC, example 16, optimizing lipid ratios for mRNA loading, and example 17, optimizing lipid ratios for plasmid loading.

Preparation of C12-200 LPMP/mRNA

This example describes LPMP formulations formulated with C12-200 as ionizable lipids, sterols, and PEG lipids, encapsulated with mRNA (e.g., 2:1 eGFP: EPO).

LNP formulations. As shown in table 2, LNP (lipid nanoparticle) formulations were prepared as controls such that the molar ratio of ionizable lipid to structural lipid to sterol to PEG-lipid (C12-200 to dope to cholesterol to DMPE-PEG2 k) was 35:16:46.5:2.5, respectively. To prepare the formulation, the above lipids were dissolved in ethanol, mixed in the above molar ratio, and diluted in ethanol (organic phase) to obtain a total lipid concentration of 12.5 mM. mRNA solutions (aqueous phase) were prepared with RNAse-free water and 100 mM citrate buffer (pH 3) at a final concentration of 50 mM. The ratio of ionizable lipid of the preparation to ionizable lipid nitrogen of the mRNA to mRNA phosphate (N: P) was maintained at 15:1.

C12-200 LPMP formulations As shown in Table 2, the C12-200 recLemon LPMP formulations were prepared such that the molar ratio of ionizable lipid to natural lipid to sterol to PEG-lipid (C12-200: lemon lipid to cholesterol to DMPE-PEG2 k) was 35:50:12.5:2.5, respectively. To prepare this formulation, the above lipids were dissolved in ethanol, except for the lemon lipid was dissolved in 4:1 DMF: methanol. The lipids were then mixed in the molar ratio described above and diluted to obtain a total lipid concentration of 5.5 mM. mRNA solutions (aqueous phase) were prepared with RNAse-free water and 100 mM citrate buffer (pH 3) at a final citrate buffer concentration of 50 mM.

The lipid mixture and mRNA solution were mixed at a volume ratio of 1:3 on NanoAssemblr Ignite (Precision Nanosystems) at a total flow rate of 9 mL/min, respectively. The resulting formulation was then loaded into Slide-A-Lyzer G2 dialysis cartridges (10 k MWCO) and dialyzed at room temperature against 200 sample volumes of 1 XPBS for 2 hours with gentle agitation. The PBS solution was refreshed and the formulation was further dialyzed at 4 ℃ for at least 14 hours with gentle agitation. The dialyzed formulation was then collected and concentrated by Tangential Flow Filtration (TFF) using Sartorius VIVAFLOW < 50 > cartridges (100 k MWCO) at a fixed feed flow rate of 150 mL/min. The formulation was then concentrated further by TFF at 3000Xg centrifugation (Amicon Ultra centrifugal filter, 100k MWCO). The size, polydispersity and particle concentration of the concentrated particles were characterized using a Zetasizer Ultra (MALVERN PANALYTICAL). mRNA encapsulation efficiency was characterized by the Quant-iT riboGreen RNA assay kit (ThermoFisher Scientific). The pellet was diluted to the desired mRNA concentration to obtain the final 10% sucrose solution in PBS. The formulation was then flash frozen in liquid nitrogen.

The resulting mRNA-containing formulations are shown in table 2.

TABLE 2 characterization of typical mRNA containing LPMP formulations

Additional mRNA design for loading into LPMP/mRNA formulations

An exemplary sequence of payloads (payload) is shown in table 3.

TABLE 3 Table 3

EXAMPLE 3 design of nano-luciferase assay

This example describes the oral administration of recLemon LPMP/mRNA formulation comprising C12-200 reconstituted lemon lipids, sterols (cholesterol) and PEG lipids (DMPE-PEG 2 k) as ionizable lipids encapsulating mRNA. recLemon LPMP/mRNA preparation tested in this example was prepared according to example 2. The coding portions of the mRNAs (nLuc-FLag and secretory nLuc) used in this example were as follows:

nLuc-Flag:

N1-methyl pseudouridine (N1-methylpseudouridine)

Cap1 CleanCap AG

ORF sequence:

Secretion type nLuc:

n1-methyl pseudouridine

Cap1 CleanCap AG

ORF sequence:

In this example, the in vivo levels of nLuc-Flag and secreted nLuc and their effects on mice after oral administration of recLemon LPMP/mRNA preparation (200 μg/200 μl nLuc mRNA) were measured. The treatment schedule is shown in the following protocol.

Mice received p.o. (oral route) administration of 200 μ g nanoLuc-Flag or secreted nano-Luc mRNA, and after 6 hours, mice were IP (intraperitoneal) administered substrate nanoluminescent material, followed by IVIS Spectrum (PerkinElmer) bioluminescence and fluorescence imaging in vivo in whole body and organ IVIS. nLuc-Flag remains in the cell and is used to determine where transfection occurs following oral delivery, secretory nLuc is used to trace the trajectory of secreted proteins following oral delivery in addition to tracing transfected cells. Secretory nLuc is also used to determine if oral delivery results in systemic delivery of secreted proteins (liver expression indicates systemic delivery). The results are shown in FIGS. 1-4. Figures 1-3 are photographs showing the levels of nano-luciferase in mice orally treated with reconstituted LPMP (recPMP) derived from lemon, reconstituted LPMP using C12-200 as an ionizable lipid, and these photographs include nano-luciferase (nLuc) mRNA-nLuc-Flag (intracellular) and secreted nLuc in multiple organs of the mice (e.g., liver, stomach, colon, spleen, small intestine, mesenteric lymph node, pancreas and cecum) with an exposure time of 5 minutes. FIG. 4 further illustrates nanoLuc-Flag (intracellular) or secreted nano-Luc levels in multiple organs of mice. These figures demonstrate that nLuc-Flag and secretory nLuc are not detected in the liver (i.e. no systemic delivery), only in the gastrointestinal tract and associated lymphoid tissues. Secretory nLuc is expressed more in the colon than nLuc-Flag.

EXAMPLE 4 administration of LNP/mRNA preparation in mice

If not specified, LNP/mRNA preparations were prepared as described in example 2.

Formulation and characterization of LNP/mRNA

This example further describes the formulation of several LNMP formulated with new ionizable lipids, structural lipids (natural or synthetic), sterols, and PEG lipids to encapsulate mRNA (e.g., nLuc-Flag mRNA, CRE recombinase mRNA) for LNP/mRNA formulation.

The coding part of nLuc-Flag mRNA used in this example is as follows:

nLuc-Flag:

n1-methyl pseudouridine

Cap1 CleanCap AG

ORF sequence:

Lipid Nanoparticle (LNP) formulations were prepared according to examples 1-3, consisting of ionizable lipids: structural lipids: sterols: PEG-lipids in the given molar ratios listed in Table 4. The lipids were dissolved in ethanol. These lipids were mixed in the indicated molar ratio and diluted in ethanol (organic phase) to a total lipid concentration of 5.5 mM, an mRNA solution (aqueous phase) was prepared with RNAse-free water and 100 mM citrate buffer (pH 3), the final citrate buffer concentration being 50 mM. The ratio of ionizable lipid to mRNA N: P of the formulations was maintained at 3:1 to 15:1 (Table 4).

TABLE 4 exemplary mRNA-LNP formulations with novel ionizable lipids

Administration and harvesting of mice

The formulations provided in Table 4 were tested using 7-8 week old C57BL/6 or TdTomato mice. Mice were obtained from Jackson laboratories and adapted for at least one week prior to handling.

In one experiment using C57BL/6 mice, jejunal delivery and oral gavage (PO) delivery were compared using lipid nanoparticles. Figures 5A-5F show the average emittance in liver, spleen, pancreas, MLN, gastrointestinal tract (GI) and inguinal lymph nodes of mice 24 hours after oral (PO) or jejunal (IJ) delivery 2243 LNP (nLuc-Flag mRNA, IJ-10 μg, PO-200 μg) (n=5/group) compared to untreated mice (n=2). The results indicate that while the IJ delivery pathway produces higher emittance at multiple locations, the PO delivery pathway is sufficient to achieve delivery of mRNA compositions and results in high biodistribution at immune locations such as MLN.

In a separate study TdTomato mice were used to determine the transfection of the gastrointestinal tract. Briefly, two TdTomato mice were given 2243 LNP (nLuc-Flag: CRE recombinase mRNA,15 μg) jejunally, and after 48 hours, the Small Intestine (SI) of the mice was imaged using IVIS. The region displaying nano-luciferase expression (i.e., the region transfected with particles) was further visualized by microscopy to identify TdTomato expression. The results are shown in FIGS. 6A-6D. Fig. 6A shows SI sections of untreated tdmamato mice, indicating baseline levels. FIG. 6B shows SI representative sections of mice given 2243 LNP (nLucFlag: CRE mRNA,15 μg) in the jejunum 48 hours prior to harvest. Fig. 6C highlights the pi spot previously shown in fig. 6B. Fig. 6D highlights the fluff previously shown in fig. 6B. These images demonstrate that lipid nanoparticles formulated with novel ionizable lipids that mimic oral delivery (e.g., IJ) result in transfection of intestinal villi and cells within the peyer's patch in the small intestine.

EXAMPLE 5 intravenous delivery of Polynucleotide encoding antibody

If not specified, a formulation was prepared as described in example 2.

The coding part of the anti-TNF heavy chain mRNA is as follows:

the coding part of the anti-TNF light chain mRNA is as follows:

The coding part of the anti-PCSK 9 heavy chain mRNA (from Vernal) used in this example is as follows:

The coding part of the anti-PCSK 9 light chain mRNA (from Vernal) used in this example is as follows:

another anti-PCSK 9 heavy chain mRNA (from Albevron) was as follows:

another anti-PCSK 9 light chain mRNA (from Albevron) was as follows:

For both heavy chain coding sequences, the encoded amino acid sequences are as follows:

For both light chain coding sequences, the encoded amino acid sequences are as follows:

formulation and characterization

This example further describes lemon LPMP or LNP formulated with ionizable lipids, sterols, and PEG lipids to encapsulate exemplary mRNA encoding antibodies, for LPMP/mRNA formulation (e.g., PCSK9 (1:1 hc: lc)) or for LNP/mRNA (e.g., TNF).

Lipid Nanoparticle (LNP) formulations were prepared according to example 2, consisting of ionizable lipids (2243): structural lipids (DSPC): sterols (plant cholesterol): PEG-lipids (DMG-PEG 2 k) in a given molar ratio of 50:10:38.5:1.5. The lipids were dissolved in ethanol. These lipids were mixed in the indicated molar ratio and diluted in ethanol (organic phase) to a total lipid concentration of 12.5 mM, an mRNA solution (aqueous phase) was prepared with RNAse-free water and 100 mM citrate buffer (pH 3), the final citrate buffer concentration being 50 mM. The ratio of ionizable lipid to mRNA N: P of the preparation was maintained at 3:1.

An exemplary lemon LPMP composition (recLemon LPMP) was prepared according to example 2, consisting of ionizable lipid (2272) to lemon lipid to cholesterol to DMG-PEG2k in a given molar ratio of 35:20:42.5:2.5. The lipids were dissolved in ethanol. The formulation was then treated as above.

The lipid mixture and mRNA solution (anti-PCSK 9 antibody mRNA or anti-tnfα antibody mRNA) were mixed at a volume ratio of 1:3 on NANOASSEMBLR cubic IGNITE ^TM (Precision Nanosystems) at a total flow rate of 12 mL/min, respectively. The resulting formulation was then loaded into Slide-A-Lyzer G2 dialysis cartridges (10 k MWCO) and dialyzed against 1 XPBS for 2 hours at room temperature. PBS was refreshed and the formulation was further dialyzed at 4 ℃ for at least 14 hours with gentle agitation. The dialyzed formulation was then collected and concentrated by Tangential Flow Filtration (TFF) using Sartorius VIVAFLOW < 50 > cartridges (100 k MWCO) at a fixed feed flow rate of 100 mL/min. The TFF concentrate formulation was then further concentrated by centrifugation at 3000Xg using an AMICON Ultra centrifuge filter (100 k MWCO). The size, polydispersity and particle concentration of the concentrated formulations were characterized using a Zetasizer Ultra (MALVERN PANALYTICAL) and mRNA encapsulation efficiency was characterized using a QUANT-IT ^TM RIBOGREEN cube RNA assay kit (ThermoFisher Scientific).

Intravenous antibody administration with LNP/mRNA formulations

Preliminary experiments were performed to determine the function of intravenous antibody administration.

TABLE 5 Experimental outline

Bl6 mice were obtained from the Jackson laboratory and were acclimatized for one week prior to the procedure. Following adaptation, mice were dosed according to table 5. On day 0, mice were started with 0% or 2% Dextran Sodium Sulfate (DSS) to induce intestinal inflammation, which is commonly used as a model of colitis. Blood and stool samples were collected on the third day. On day 4, the appropriate experimental group was given 0.6 mg/kg 2243 LNP/anti-TNF mRNA intravenously. Blood and stool samples were collected on days 5 and 7.

FIG. 7 depicts the concentration of anti-TNFα antibody levels in 24h plasma following single intravenous administration of either mice administered 0% DSS (2243 LNP/anti-TNFα mRNA,0.6 mg/kg) or mice administered 2% DSS (2243 LNP/anti-TNFα mRNA,0.6 mg/kg) with single intravenous administration of LNP/mRNA formulation. The control was plasma of untreated mice (0% DSS and no drug). N=5/group. The results indicate that anti-TNF is detectable in mice 24 hours after administration.

FIG. 8A shows TNFα concentrations in feces three days after administration of mice administered 2% DSS or mice administered 2% DSS and single intravenous administration of LNP/mRNA formulation (2243 LNP/anti-TNFα mRNA,0.6 mg/kg). The control was faeces of untreated mice (0% DSS and no drug administration). N=5/group. Fig. 8B shows calprotectin concentrations in feces of mice given 2% DSS and single intravenous administration of LNP/mRNA formulation (2243 LNP/anti-tnfa mRNA,0.6 mg/kg) before dosing (3 days after DSS start), 24h after dosing (5 days after DSS start), and mice given 2% DSS and single intravenous administration of LNP/mRNA formulation (2243 LNP/anti-tnfa mRNA,0.6 mg/kg) at 3 days after dosing (7 days after DSS start). The control was faeces of untreated mice (0% DSS and no LNP 2243/anti tnfα mRNA administered). N=5/group. The results indicate that single intravenous administration of LNP (encapsulated anti-tnfa mRNA) formulated with ionizable lipid 2243 reduced inflammatory measurements in a colitis model, measured by fecal TNF and calprotectin levels. Fecal calprotectin results observed at 24h post-dose persisted for 3 days post-dose.

Intravenous antibody administration with LNMP/mRNA formulations

Mice of 6-8 weeks of age were obtained from Jackson laboratories and adapted for at least one week prior to handling. In one experiment, 100 μ L LPMP/mRNA preparation (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg) was administered intravenously to mice. Tail blood on days 0, 1, 3, 7 and 14 was collected, treated and tested for anti-PCSK 9 concentration. The results are shown in FIG. 9.

Fig. 9 shows the antibody concentrations (huIgG, ng/mL) n=5 measured in mouse plasma at days 0,1, 3, 7 and 14 after single intravenous administration of LPMP/mRNA formulation (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg) using ionizable lipid 2272. The results indicate that antibodies can be detected systemically up to 14 days after administration.

In a separate experiment, mice were dosed intravenously with LPMP/mRNA preparation of ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg), 24h post-dose, and plasma, colon and cecum and Mesenteric Lymph Node (MLN) samples were collected. anti-PCSK 9 concentration (ng/mL) in a given sample is measured. The results are shown in FIGS. 10A-10B.

Fig. 10A shows the antibody concentration (huIgG, ng/mL) n=10 measured in plasma of 24 h mice after a single intravenous administration of LPMP/mRNA formulation using ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg). PBS was used as a control (n=1). Fig. 10B shows the antibody concentration (huIgG, ng/mL) n=10 measured in MLN and colon/cecum of 24 h mice after a single intravenous administration of LPMP/mRNA formulation using ionizable lipid 2272 (recLemon LPMP 2272/anti-PCSK 9 mRNA,0.3 mg/kg). PBS was used as a control (n=1). The results indicate that the antibodies were detectable in both the whole body as well as in the lymph nodes and colon/cecum compared to control mice given PBS.

EXAMPLE 6 administration of LNMP/mRNA preparation in mice

If not specified, LNMP/mRNA preparations were prepared as described in example 2.

Formulation and characterization of LNMP/mRNA

This example further describes the formulation of several LNMPs formulated with new ionizable lipids, structural lipids (natural or synthetic), sterols, and PEG lipids to encapsulate mRNA (e.g., anti-PCSK 9 mRNA) for use in LNMP/mRNA formulations. LNMP compositions in which the natural lipid was extracted from a plant source (i.e., lemon in this example) are labeled LPMP.

another anti-PCSK 9 heavy chain mRNA (from Albevron) was as follows:

another anti-PCSK 9 light chain mRNA (from Albevron) was as follows:

The exemplary lemon LPMP composition was formulated in accordance with examples 1-3, consisting of the ionizable lipids: lemon lipids: sterols: PEG-lipids in the given molar ratios listed in table 6. The lipids were dissolved in ethanol. These lipids were mixed in the indicated molar ratio and diluted in ethanol (organic phase) to a total lipid concentration of 5.5 mM, an mRNA solution (aqueous phase) was prepared with RNAse-free water and 100 mM citrate buffer (pH 3), the final citrate buffer concentration being 50 mM. The ratio of ionizable lipid to mRNA N: P of the formulations was maintained at 3:1 to 15:1 (Table 6).

The lipid mixture and mRNA solution were mixed at a volume ratio of 1:3 on NANOASSEMBLR cubic IGNITE ^TM (Precision Nanosystems) at a total flow rate of 14 mL/min, respectively. The resulting formulation was then loaded into Slide-A-Lyzer G2 dialysis cartridges (10 k MWCO) and dialyzed against 1 XPBS for 2 hours at room temperature. PBS was refreshed and the formulation was further dialyzed at 4 ℃ for at least 14 hours with gentle agitation. The dialyzed formulation was then collected and concentrated by centrifugation at 2000Xg using an AMICON Ultra centrifuge filter (100 k MWCO). The size, polydispersity and particle concentration of the concentrated formulations were characterized using a Zetasizer Ultra (MALVERN PANALYTICAL) and mRNA encapsulation efficiency was characterized using a QUANT-IT ^TM RIBOGREEN cube RNA assay kit (ThermoFisher Scientific).

TABLE 6 LPMP formulations with novel ionizable lipids

*1:1 HC: LC represents 1:1 heavy chain: light chain

Administration and Collection in mice

The formulations provided in Table 6 were tested using 7-8 week old C57BL/6 mice with PBS as a control. Mice were obtained from Jackson laboratories and adapted for at least one week prior to handling.

In one experiment, 19 mice given jejunally were used to measure antibody concentration during 3 independent experiments. Figures 11A-11D show the concentration of antibodies (huIgG, ng/mL) in colon/cecum content, mesenteric Lymph Node (MLN), small Intestine (SI) content and plasma of mice 24 hours after jejunal delivery 2272 LPMP (anti-PCSK 9 mRNA,65-75 μg). This experiment was an n=19/3 independent experiment compared to PBS (n=16/3 independent experiment). Statistically identified outliers are deleted. The results indicate that jejunal delivery (an alternative mode of oral delivery) is sufficient to deliver the mRNA therapeutic composition to the mice.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, such description and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety. Other embodiments are within the scope of the following claims.

Claims

1. A method for delivering an RNA composition to a subject to produce a protein or peptide in the subject, comprising:

An RNA composition comprising a polynucleotide encoding a protein or peptide is administered to the subject, the polynucleotide being formulated in the following structure:

(a) Multiple lipid nanoparticles (LNPs) containing synthetic structural lipids and ionizable lipids, or

(b) Lipid reconstructed natural messenger packages (LNMPs) containing natural lipids and ionizable lipids.

The ionizable lipids described herein have two or more of the following characteristics:

(i) at least one ionizable amine functional group;

(iii) pKa is approximately 4.5 to approximately 7.5;

(iv) Ionizable amine functional groups and heteroorganic functional groups are separated by chains of at least two atoms; and

(v) The N:P ratio is at least 3.

2. The method of claim 1, wherein the RNA composition is administered orally or intestinally.

3. The method of claim 1, wherein the RNA composition is administered systemically.

4. The method of claim 1, wherein the polynucleotide is mRNA or circRNA, and optionally wherein the mRNA or circRNA is derived from...

(a) DNA molecule; or

(b) RNA molecule in which T is replaced by U.

5. The method of claim 1, wherein the protein or peptide comprises an antibody.

6. The method of claim 5, wherein the antibody is a therapeutic agent, a TNF inhibitor, or a PCSK9 inhibitor.

7. The method of claim 1, wherein the in vivo production of the protein or peptide occurs in the stomach, small intestine, mesenteric lymph nodes, pancreas, colon, cecum and/or spleen of the subject.

8. The method of claim 1, wherein administration of the RNA composition results in: at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration, the expression of the protein or peptide encoded by the polynucleotide is detectable in one or more organs of the subject, and

The organs are distributed along the transport pathway of the digestive tract, and the RNA composition reaches the organs via the lymphatic transport system.

9. The method of claim 1, wherein, at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration, the protein or peptide encoded by the polynucleotide is detectable in the mesenteric lymph nodes, pancreas, stomach, colon, small intestine, spleen, villi, and/or Pell's spots of the subject.

10. The method of claim 1, wherein the protein or peptide encoded by the polynucleotide is detectable in the liver of the subject at least about 6 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, or about 96 hours after administration.

11. A method for treating diseases or disorders related to the gastrointestinal tract, stomach, small or large intestine, mesenteric lymph nodes, pancreas, colon or rectum, cecum, and/or spleen, comprising:

An RNA composition comprising a polynucleotide encoding one or more polypeptides, administered orally or intestinally, wherein the polynucleotide is formulated in the following structure:

(i) at least one ionizable amine functional group;

(iii) pKa is approximately 4.5 to approximately 7.5;

(v) The N:P ratio is at least 3.

12. The method of claim 11, wherein the disease or disorder is pancreatitis, IBD, Crohn's disease, colorectal cancer, or ulcerative colitis.

13. The method of any one of claims 1-12, wherein the RNA composition is formulated within (a) a plurality of lipid nanoparticles (LNPs) comprising synthetic structural lipids and ionizable lipids.

14. The method of any one of claims 1-12, wherein the RNA composition is formulated in (b) a lipid reconstituted natural messenger package (LNMP) comprising natural lipids and ionizable lipids.

15. The method of any one of the preceding claims, wherein the RNA composition is administered to the subject in a delayed-release drug formulation comprising: (a) a therapeutically effective amount of the polynucleotide; (b) a bile salt or bile acid; and (c) at least one surfactant selected from hydrophilic surfactants, lipophilic surfactants, and mixtures thereof.

16. The method of claim 15, wherein the RNA composition is administered in capsule form.

17. The method of claim 16, wherein the capsule is a starch capsule, a cellulose capsule, a hard gelatin capsule, or a soft gelatin capsule.

18. The method of claim 15, wherein the RNA composition is administered in the form of a tablet or capsule.

19. The method of any one of claims 16-18, wherein the capsule, tablet, or capsule-shaped tablet comprises an enteric coating.

20. The method of claim 15, wherein the RNA composition is applied in the form of a plurality of particles, microparticles, beads, pellets or mixtures thereof.

21. The method of any one of the preceding claims, wherein the ionizable lipid is selected from the group consisting of: 1,1’-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azadiyl)bis(dodecyl-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, lipid 5, SM-102 (lipid H) and ALC-315.

22. The method of any one of claims 1-20, wherein the ionizable lipid is selected from one of the group consisting of:

i) Formula The compound, its pharmaceutically acceptable salt, or a stereoisomer of any of the foregoing,

in:

Each A is independently a _C1 - _C16 branched or unbranched alkyl or _C1 - _C16 branched or unbranched alkenyl, optionally substituted with a heteroatom or substituted with OH, SH or halogen;

Each B is independently a _C1 - _C16 branched or unbranched alkyl or _C1 - _C16 branched or unbranched alkenyl, optionally substituted with a heteroatom or substituted with OH, SH or halogen;

Each X is independently a biodegradable portion; and

W is

;or ,

in,

_R5 is OH, SH, or NR ₁₀ _R11 ;

Each R ₆ is independently hydrogen, _C1 - _C3 branched or unbranched alkyl, _C2 - _C3 branched or unbranched alkenyl, or cycloalkyl;

Each _R7 and each _R8 is independently hydrogen, _C1 - _C3 branched or unbranched alkyl, _C2 - _C3 branched or unbranched alkenyl, halogen, OH, SH or _NR10 _R11 , wherein each _R10 and _R11 is independently H, _C1 - _C3 alkyl, or _R10 and _R11 together form a heterocycle;

Each s is independently 1, 2, 3, 4 or 5;

Each u is independently 1, 2, 3, 4 or 5;

t is 1, 2, 3, 4, or 5;

Each Z is independently absent, O, S, or NR ₁₂ , wherein R ₁₂ is H, _C1 - _C7 branched or unbranched alkyl, or _C2 - _C7 branched or unbranched alkenyl; and

Q is O, S or NR ₁₃ , wherein each R ₁₃ is H or _C1 - _C5 alkyl;

ii) Formula The compound, its pharmaceutically acceptable salt, or a stereoisomer of any of the foregoing, wherein:

It is either a cyclic or heterocyclic part;

Y is alkyl, hydroxyl, hydroxyalkyl, or... ;

A is absent, -O-, -N( ^R7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N( ^R7 )C(O)-, -C(O)N( ^R7 )-, -N( ^R7 )C(O)N( ^R7 )-, -S-, -SS-, or a divalent heterocycle;

Each of X and Z is independently absent, -O-, -CO-, -N( ^R7 )-, -O-alkylene-, -alkylene-O-, -OC(O)-, -C(O)O-, -N( ^R7 )C(O)-, -C(O)N( ^R7 )- or -S-;

Each M is an independent biodegradable portion;

Each of _R30 , _R40 , _R50 , _R60 , _R70 , _R80 , _R90 , _R100 , _R110 and _R120 is independently H, _C1 - _C16 branched or unbranched alkyl or _C1 - _C16 branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl;

Each of l and m is an integer from 1 to 10;

t1 is an integer from 0 to 10; and

W is a hydroxyl group, a substituted or unsubstituted hydroxyalkyl group, a substituted or unsubstituted amino group, a substituted or unsubstituted aminocarbonyl group, or a substituted or unsubstituted heterocyclic or heteroaryl group; and

equation iii) or The compound, its pharmaceutically acceptable salt, or a stereoisomer of any of the foregoing, wherein:

_R20 and _R30 are each independently H, _C1 - _C5 branched or unbranched alkyl, or _C2 - _C5 branched or unbranched alkenyl, or _R20 and _R30 together with the adjacent N atom to form a 3 to 7-membered ring, optionally substituted with ^Ra ;

^Ra can be H, _C1 - _C3 branched or unbranched alkyl, _C2 - _C3 branched or unbranched alkenyl, halogen, OH or SH;

Each _R1 and each _R2 is independently H, _C1 - _C3 branched or unbranched alkyl, _C2 - _C3 branched or unbranched alkenyl, OH, halogen, SH, or _NR10R11 , _or

_R1 and _R2 together form a ring;

Each _R10 and _R11 is independently H, _C1 - _C3 branched or unbranched alkyl, _C2 - _C3 branched or unbranched alkenyl, or _R10 and _R11 together form a heterocycle;

n is 0, 1, 2, 3 or 4;

Y is either O or S;

Z is absent, O, S or N (R ₁₂ ), wherein each R ₁₂ is independently H, C ₁ -C ₇ branched or unbranched alkyl, or C ₂ -C ₇ branched or unbranched alkenyl, provided that when Z is present, the adjacent R ₁ and R ₂ cannot be OH, NR ₁₀ R ₁₁ or SH.

v can be 0, 1, 2, 3, or 4;

y is 0, 1, 2, 3 or 4;

Each A is independently a _C1 - _C16 branched or unbranched alkyl or a _C2 - _C16 branched or unbranched alkenyl, optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH or halogen;

Each B is independently a _C1 - _C16 branched or unbranched alkyl group, or a _C2 - _C16 branched or unbranched alkenyl group, optionally interrupted by one or more heteroatoms or optionally substituted by OH, SH, or a halogen; and

Each X is independently a biodegradable portion; and

iv) A lipid comprising at least one head group and at least one tail group of formula (TI) or (TI’), a pharmaceutically acceptable salt thereof, or a stereoisomer of any of the foregoing:

or ,

in:

Each of E is independently -OC(O)-, -C(O)O-, -N( ^R7 )C(O)-, -C(O)N( ^R7 )-, -C( _OR13 )-O-, -C(O)O( _CH2 ) _r- , -C(O)N( ^R7 )( _CH2 ) _r- , -SS- or -C( _OR13 )-O-( _CH2 ) _r- , wherein each ^R7 is independently H, alkyl, alkenyl, cycloalkyl, hydroxyalkyl or aminoalkyl;

R ₁₃ is a branched or unbranched _C3 - _C10 alkyl group;

r is 1, 2, 3, 4 or 5;

Ra ^is independently _C1 - _C5 alkyl, _C2 - _C5 alkenyl or _C2 - _C5 ynyl;

u1 and u2 are each independently 0, 1, 2, 3, 4, 5, 6 or 7;

R ^t is independently H, C _1 -C _16 branched or unbranched alkyl or C ₁ -C ₁₆ branched or unbranched alkenyl, optionally interrupted by heteroatoms or substituted by OH, SH or halogen or cycloalkyl or substituted cycloalkyl.

This represents the bond connecting the tail group and the head group; and

The pKa of the lipid is approximately 4 to approximately 8.

23. The method of claim 22, wherein the ionizable lipid is a compound in Table I, Table II, Table III or Table IV.

24. The method of claim 23, wherein the ionizable lipid is

or

.

25. The method of claim 14, wherein the natural lipids of the LNMP are extracted from lemons or algae.

26. The method of claim 13 or 14, wherein the LNMP or the LNP composition further comprises a sterol and a polyethylene glycol (PEG) lipid conjugate.

27. The method of claim 26, wherein the PEG-lipid conjugate is PEG-DMG or PEG-PE.

28. The method of claim 27, wherein the PEG-DMG is PEG2000-DMG or PEG2000-PE.

29. The method of claim 26, wherein the LNMP comprises:

The ionizable lipids comprised approximately 20 mol% to approximately 50 mol%.

The natural lipids comprise approximately 5 mol% to approximately 60 mol%.

The sterols, in amounts of about 7 mol% to about 50 mol%, and

The polyethylene glycol (PEG)-lipid conjugate comprises about 0.5 mol% to about 3 mol%.

30. The method of claim 29, wherein the LNMP comprises an ionizable lipid: natural lipid: sterol: PEG-lipid in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:30:32.5:2.5, about 35:16:46.5:2.5, about 35:25:37.5:2.5, about 35:40:22.5:2.5, about 45:10:43.5:1.5, about 50:20:28.5:1.5, or about 50:10:38.5:1.5.

31. The method of claim 13, wherein the synthetic structural lipid of the LNP composition is a phospholipid selected from the group consisting of: lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, lysophosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, diceryl phosphate, distearate, dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyl oil Acylphosphatidylcholine (POPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), dioleoyl phosphatidylethanolamine 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid ester (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, ditransoleoyl phosphatidylethanolamine (DEPE), stearoyl oleoyl phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof.

32. The method of claim 26, wherein the LNP composition comprises:

The ionizable lipids comprised approximately 20 mol% to approximately 50 mol%.

The synthetic structural lipids comprise approximately 5 mol% to approximately 60 mol% of the total amount.

The sterols, in amounts of about 7 mol% to about 50 mol%, and

33. The method of claim 32, wherein the LNP composition comprises an ionizable lipid:synthetic structural lipid:sterol:PEG-lipid in a molar ratio of about 35:50:12.5:2.5, about 35:20:42.5:2.5, about 35:30:32.5:2.5, about 35:16:46.5:2.5, about 35:25:37.5:2.5, about 35:40:22.5:2.5, about 45:10:43.5:1.5, about 50:20:28.5:1.5, or about 50:10:38.5:1.5.

34. The method of claim 1, wherein the total lipid:polynucleotide weight ratio of the RNA composition ranges from about 50:1 to about 10:1.

35. The method of claim 34, wherein the total lipid:polynucleotide weight ratio of the RNA composition ranges from about 40:1 to about 28:1.

36. The method of claim 34, wherein the total lipid:polynucleotide weight ratio of the RNA composition ranges from about 37:1 to about 33:1.

37. An oral vaccine composition comprising an RNA composition containing a polynucleotide encoding one or more polypeptides, said polynucleotide being formulated in the following structure:

(a) Multiple lipid nanoparticles (LNPs) containing synthetically structured lipids and ionizable lipids, or

(i) at least one ionizable functional group (amine);

(iii) pKa is approximately 4.5 to approximately 7.5;

(v) The N:P ratio is at least 3, wherein the RNA composition is formulated in an oral dosage form.

38. The oral vaccine composition of claim 37, wherein the polypeptide is an antigenic polypeptide derived from an infectious pathogen causing viral or bacterial infection, optionally wherein the antigenic polypeptide is a coronavirus.

39. The oral vaccine composition of claim 37, wherein the polypeptide is a tumor antigen polypeptide.