CN120153078A

CN120153078A - RNA molecules encoding RSV-F and vaccines containing the same

Info

Publication number: CN120153078A
Application number: CN202380075365.8A
Authority: CN
Inventors: 陈伟; F·M·迪亚兹; K·A·斯旺森
Original assignee: Pfizer Corp SRL
Current assignee: Pfizer Corp SRL
Priority date: 2022-10-27
Filing date: 2023-10-26
Publication date: 2025-06-13
Also published as: CO2025005088A2; AU2023369585A1; KR20250096808A; MX2025004821A; TWI888970B; TW202424187A; IL320459A; WO2024089633A1; PE20251592A1; EP4608440A1

Abstract

The present invention relates to RNA molecules encoding Respiratory Syncytial Virus (RSV). The invention further relates to compositions comprising said RNA molecules (RNA-LNP) formulated in lipid nanoparticles. The invention further relates to the use of said RNA molecule, said RNA-LNP and said composition for the treatment and/or prevention of RSV infection induced acute respiratory disorders including pneumonia and bronchitis.

Description

RNA molecules encoding RSV-F and vaccines containing the same

Cross-reference to related applications

U.S. provisional application No. 63/585,254 to 26, 2023, 9, and 2022, 10, 27 claims the benefit of U.S. provisional application No. 63/381,238. The entire contents of each of the foregoing applications are incorporated herein by reference.

[ Reference to the sequence Listing ]

The present application is electronically applied via an EFS network and includes a sequence listing that is electronically submitted in an xml format. An xml archive contains a Sequence listing named "PC072895A Sequence listing. Xml", which was created at 2023, 9, 25 and has a size of 145KB. The sequence listing contained in this xml file is part of the specification and is incorporated by reference herein in its entirety.

[ PRIOR ART ]

Respiratory syncytial virus (Respiratory syncytial virus; RSV) is a respiratory virus that infects the lung and respiratory tract. RSV is a major cause of severe viral lower respiratory tract disorders in infants worldwide and in elderly people. Two RSV protein subunit vaccines, ABRYSVO (Pfizer) and AREXVY (GSK), were approved in 2023. However, no RNA vaccine has been approved for the prevention of RSV infection.

RSV is a member of the pneumoviridae family. The genome consists of single-stranded, antisense RNA molecules encoding 11 proteins (including nine structural proteins (three glycoproteins and six internal proteins) and two non-structural proteins). The structural proteins comprise three transmembrane surface glycoproteins, namely attachment protein G, fusion protein F and hydrophobic small SH protein. There are two RSV subtypes, a and B. It differs mainly in G glycoprotein, while the sequence of F glycoprotein between the two subtypes is more conserved.

Mature F glycoprotein has three general domains, extracellular Domain (ED), transmembrane domain (TM) and Cytoplasmic Tail (CT). CT contains a single palmitoylated cysteine residue.

The F glycoprotein of human RSV is initially translated from mRNA into a single 574 amino acid polypeptide precursor (termed "F0" or "F0 precursor") that contains a signal peptide sequence (amino acids 1-25) at the N-terminus. After translation, the signal peptide is removed in the endoplasmic reticulum by a signal peptidase. The remainder of the F0 precursor (i.e., residues 26-574) can be cleaved at two polybasic sites (a.a. 109/110 and 136/137) by cellular proteases, particularly furin (furin), removing the 27 amino acid insertion sequence termed pep27 (amino acids 110-136) and producing two linked fragments termed F1 (C-terminal portion; amino acids 137-574) and F2 (N-terminal portion; amino acids 26-109). F1 contains a hydrophobic fusion peptide at its N-terminus, and two heptad (heptad) repeat regions (HRA and HRB). HRA is near the fusion peptide and HRB is near the TM domain. The F1 and F2 fragments are linked together via two disulfide bonds. One of the uncleaved F0 protein or the F1-F2 heterodimer without the signal peptide sequence may form an RSV F protomer. Three such protomers assemble to form a final RSV F protein complex, which is a homotrimer of the three protomers.

The F proteins of subtypes a and B are about 90% identical in amino acid sequence. An example sequence for a subtype A F0 precursor polypeptide is provided in SEQ ID NO. 1 (strain A2; genBank GI:138251;Swiss Prot P03420), and an example sequence for a subtype B F0 precursor polypeptide is provided in SEQ ID NO. 2 (strain 18537; genBankGI:138250;Swiss Prot P13843). SEQ ID NO. 1 and SEQ ID NO. 2 are all 574 amino acid sequences. The signal peptide sequences of SEQ ID NO. 1 and SEQ ID NO. 2 have also been reported as amino acids 1 to 25 (GenBank and UniProt). In both sequences, the TM domain is approximately amino acids 530-550, but is alternatively reported as 525-548. The cytoplasmic tail starts at amino acid 548 or 550 and at amino acid 574, wherein the palmitoylated cysteine residue is located at amino acid 550.

The RSV F protein is the primary antigen of the search for RSV vaccines. The RSV F protein trimer mediates fusion between the viral particle membrane and the host cell membrane and also promotes syncytia formation. In the viral particles prior to fusion with the membrane of the host cell, the largest population of F molecules forms a lollipop-like structure, wherein the TM domain is anchored in the viral envelope [ Dormitzer, p.r., grandi, g., rappuoli, r., nature Reviews Microbiol,10,807,2012 ]. This configuration is referred to as a pre-fusion configuration. Pre-fusion RSV F is recognized by monoclonal antibodies (mAbs) D25, AM22 and MPE8, and there is no distinction between oligomeric states. Pre-fusion F trimers are specifically recognized by mAb AM14 [ Gilman MS, moin SM, mas V et al, PLoS Pathogens,11 (7), 2015]. During RSV entry into cells, the F protein rearranges from a pre-fusion state (which may be referred to herein as "pre-F") to a post-fusion state ("post-F") via an intermediate extension structure. During this rearrangement, the C-terminal coiled-coil of the pre-fusion molecule dissociates into its three constituent chains, which then wind around the globular head and join the three additional coils to form a post-fusion six-helix bundle. If the pre-fusion RSV F trimer is subjected to increasingly severe chemical or physical conditions (such as high temperatures), it undergoes structural changes. Initially, the trimeric structure is lost (at least locally within the molecule) and then rearranged into a post-fusion form, and then the domain is denatured.

In order to prevent viral entry, the F-specific neutralizing antibodies may have to bind to the pre-fusion conformation or possibly the extended intermediate of F on the viral particle before the viral envelope fuses with the cell membrane. Thus, the pre-fusion form of the F protein is considered to be a preferred conformation [Ngwuta,J.O.,Chen,M.,Modjarrad,K.,Joyce,M.G.,Kanekiyo,M.,Kumar,A.,Yassine,H.M.,Moin,S.M.,Killikelly,A.M.,Chuang,G.Y.,Druz,A.,Georgiev,I.S.,Rundlet,E.J.,Sastry,M.,Stewart-Jones,G.B.,Yang.Y.,Zhang,B.,Nason,M.C.,Capella,C.,Peeples,M.,Ledgerwood,J.E.,Mclellan,J.S.,Kwong,P.D.,Graham,B.S.,Science Translat.Med.,14,7,309(2015)]. as the desired vaccine antigen, which is readily converted to the post-fusion form [ McLellan JS, chen M, leung S et al Structure of RSV fusion glycoprotein trimer bound to a pre-fusion-specific neutralizing antibody.Science 340,1113-1117(2013);Chaiwatpongsakorn,S.,Epand,R.F.,Collins,P.L.,Epand R.M.,Peeples,M.E.,J Virol.85(8):3968-77(2011);Yunus,A.S.,Jackson T.P.,Crisafi,K.,Burimski,I.,Kilgore,N.R.,Zoumplis,D.,Allaway,G.P.,Wild,C.T.,Salzwedel,K.Virology.2010 for 20 months; 396 (2): 226-37] upon extraction from the membrane with a surfactant such as Triton (Triton) X-100, triton X-114, NP-40, brij-35, brij-58, tween (Tween) 20, tween 80, octyl glucoside, octyl thioglucoside, SDS, CHAPS, CHAPSO), or upon expression as an ectodomain, physical or chemical stress, or upon storage. Thus, preparing pre-fusion F as a vaccine antigen remains a challenge. Because of neutralization and protective antibody function by interfering virus entry, F antigens that do not elicit pre-fusion specific antibodies are not expected to be as effective as F antigens that elicit pre-fusion specific antibodies. Thus, it is believed that there is a greater need to utilize F protein vaccines that contain F protein immunogens in a pre-fusion form. Mutants of RSV F protein have been provided to increase pre-fusion stability (see, e.g., PCT application No. WO 2017/109629) and are promising vaccine candidates.

RSV vaccines incorporating F protein antigens have been developed. Clinical studies have shown that some vaccine candidates based on the F protein subunit are safe and immunogenic, but there is a need for improvement in protective efficacy and protective persistence.

Thus, there is a need for improved immunogenic compositions to protect against RSV infection.

[ Invention ]

The present invention provides an unmet need for improved immunogenic compositions against RSV infection, particularly as provided herein. In one embodiment, the invention provides immunogenic compositions and methods for preventing, treating, or ameliorating an infection, disease, or condition in an individual comprising administering an RNA molecule, such as an immunogenic RNA polynucleotide encoding an amino acid sequence (e.g., an immunogenic antigen comprising a Respiratory Syncytial Virus (RSV) protein, an immunogenic variant thereof, or an immunogenic fragment of an RSV protein or an immunogenic variant thereof, such as an antigenic peptide or protein). Thus, the immunogenic antigen comprises an epitope of RSV protein for inducing an immune response against RSV in an individual. RNA polynucleotides encoding immunogenic antigens are administered to provide (after expression of the polynucleotide by an appropriate target cell) antigens for inducing (e.g., stimulating, initiating, and/or amplifying) an immune response (e.g., antibodies and/or immune effector cells). In one embodiment, the immune response induced according to the invention is both a B cell mediated immune response (e.g., an antibody mediated immune response) and a T cell mediated immune response. In one embodiment, the immune response is an anti-RSV immune response.

The immunogenic compositions described herein comprise an RNA molecule comprising RNA (as an active ingredient) that can be translated into one or more proteins in a recipient's cell. In addition to the wild-type, codon-optimized or mutant sequences encoding the antigen sequences, the RNA molecules may contain one or more structural components (5 ' cap, 5' utr, subgenomic promoter, 3' utr, poly a tail) optimized for maximum efficacy of the RNA in terms of stability and translation efficiency. In one embodiment, the RNA molecule contains all of these components. The RNA molecules described herein can be complexed with lipids and/or proteins to produce RNA-particles (e.g., lipid Nanoparticles (LNPs)) for administration. In one embodiment, the RNA molecules described herein are complexed with lipids to produce RNA-lipid nanoparticles (e.g., RNA-LNP) for administration. In one embodiment, the RNA molecules described herein are complexed with a protein for administration. In one embodiment, the RNA molecules described herein are complexed with lipids and proteins for administration. If a combination of different RNA molecules is used, the RNA molecules may be complexed together with the lipid and/or protein or separately complexed together with the lipid and/or protein to produce an RNA-particle for administration.

The present invention provides RNA molecules and RNA-LNPs comprising at least one open reading frame (open READING FRAME; ORF) encoding an RSV antigen. In some embodiments, the RSV antigen is an RSV polypeptide. In some embodiments, the RSV polypeptide is an RSV F protein. In some embodiments, the RSV F protein is a full-length protein, a truncated protein, a fragment or variant thereof. In some embodiments, the RSV F protein comprises at least one mutation.

The present invention provides RNA molecules and RNA-LNPs comprising at least one ORF encoding an RSV polypeptide of table 1. In some embodiments, the RSV polypeptide comprises an amino acid sequence selected from SEQ ID NOS 1-6 or 71-74. In some embodiments, the RSV polypeptides have, have at least, or at most, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity to any of the amino acid sequences of table 1 (e.g., any of SEQ ID NOs: 1-6 or 71-74). In some embodiments, the RSV polypeptide consists of any of the amino acid sequences of Table 1 (e.g., any of SEQ ID NOS: 1-6 or 71-74).

The present invention provides RNA molecules and RNA-LNPs comprising at least one ORF transcribed from at least one DNA nucleic acid of table 2. In some embodiments, the RNA molecule is transcribed from a nucleic acid sequence selected from the group consisting of SEQ ID NOS 7-10 or 59-62. In some embodiments, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having, having at least or at most 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or more identity to any of the nucleic acid sequences of Table 2 (e.g., any of SEQ ID NOS: 7-10 or 59-62). In some embodiments, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any of the nucleic acid sequences of Table 2 (e.g., any of SEQ ID NOS: 7-10 or 59-62).

The invention further provides RNA molecules and RNA-LNPs comprising at least one ORF comprising the RNA nucleic acid sequences of Table 3. In some embodiments, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOS 11-16 or 63-70. In some embodiments, the RNA molecule comprises a nucleic acid sequence having, having at least, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any of the nucleic acid sequences of Table 3 (e.g., any of SEQ ID NOS: 11-16 or 63-70). In some embodiments, the RNA molecule comprises a nucleic acid sequence consisting of any of the nucleic acid sequences of Table 3 (e.g., any of SEQ ID NOS: 11-16 or 63-70). In some embodiments, each uridine of any of SEQ ID NOs 11-16 is replaced with N1-methyl pseudouridine (ψ) (e.g., modified RNA; modRNA).

The invention further provides RNA molecules and RNA-LNPs comprising a 5 'untranslated region (5' -UTR) and/or a3 'untranslated region (3' -UTR). In some embodiments, the RNA molecule includes a 5 'untranslated region (5' -UTR). In some embodiments, the 5' UTR comprises a sequence selected from any one of SEQ ID NOs 17 to 19. In some embodiments, the 5' UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or more identity to any one of SEQ ID NOs 17-19. In some embodiments, the 5' UTR comprises a sequence selected from any one of SEQ ID NOs 17 to 19. In some embodiments, the 5' UTR comprises a sequence consisting of any one of SEQ ID NOs 17 to 19.

In some embodiments, the RNA molecules and RNA-LNPs include a 3 'untranslated region (3' -UTR). In some embodiments, the 3' UTR comprises a sequence selected from any one of SEQ ID NOS: 20-25. In some embodiments, the 3' UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or more identity to any one of SEQ ID NOs 20-25. In some embodiments, the 3' UTR comprises a sequence selected from any one of SEQ ID NOS: 20-25. In some embodiments, the 3' UTR comprises a sequence consisting of any one of SEQ ID NOs 20 to 25.

The invention further provides RNA molecules and RNA-LNPs comprising a 5' cap portion. In some embodiments, the 5 'cap moiety is (3' ome) -m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG. The invention further provides RNA molecules and RNA-LNPs comprising a 3' poly-A tail. In some embodiments, the poly A tail comprises a sequence having SEQ ID NO. 26.

In some embodiments, the RNA molecule includes a 5'utr and a 3' utr. In some embodiments, the RNA molecule includes a 5' cap, a 5' utr, and a 3' utr. In some embodiments, the RNA molecule includes a 5' cap, a 5' utr, a 3' utr, and a poly a tail. In some embodiments, the RNA molecules include a 5'utr, a 3' utr, and a poly a tail. In some embodiments, 1,2, 3 or more of the foregoing elements may be excluded from the RNA molecule. In some embodiments, each uridine of any of the 5'UTR, 3' UTR, and poly-A tail is replaced with N1-methyl pseudouridine (ψ) (e.g., modified RNA; modRNA).

In some embodiments, the multiple A tail length may contain +1/-1A. In some embodiments, the uridine is N1-methyl pseudouridine (ψ).

The present invention provides RNA molecules described in table 5. In some embodiments, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 11, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 12, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 63, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 65, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 67, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In another embodiment, the RNA molecule comprises the 5'UTR of SEQ ID NO. 18, the RSV ORF of SEQ ID NO. 69, the 3' UTR of SEQ ID NO. 21 and/or the multi-A tail of SEQ ID NO. 26. In some embodiments RSVORF further comprises a stop codon as described herein. In some embodiments, the multi-A tail length may contain +1/-1A or +2/-2A. In some embodiments, each uridine of the RNA molecule is replaced with N1-methyl pseudouridine (ψ) (e.g., modified RNA; modRNA).

The invention further provides an RNA molecule comprising at least one open reading frame produced by codon optimized DNA. In some embodiments, the open reading frame comprises at least, up to, just below, or between any two of the following (inclusive or exclusive) G/C content ：50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％ or 75%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, is or is about 50% -75%, or is about 55% -70%. In some embodiments, the G/C content is at or about 58%, at or about 66%, or at or about 62%.

The invention further provides RNA molecules comprising stabilized RNA. The invention further provides RNA molecules comprising RNA having at least one modified nucleotide (e.g., modified RNA; modRNA). In some embodiments, the modified nucleotide is pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine, or 2' -O-methyl uridine. In some embodiments, the modified nucleotide is N1-methyl pseudouridine (ψ). In some embodiments, 1,2, 3, 4, 5 or more of the foregoing modified nucleotides may be excluded from the RNA molecule.

The invention further provides RNA molecules, which are messenger RNAs (mrnas) or self-replicating RNAs. In some embodiments, the RNA is mRNA.

The invention further provides immunogenic compositions comprising the RNA molecules described herein. RNA molecules can be formulated in such immunogenic compositions, encapsulated therein, complexed with Lipid Nanoparticles (LNP) therein, bound to LNP, or adsorbed onto LNP (e.g., RSV RNA-LNP). In some embodiments, the lipid nanoparticle includes at least one of a cationic lipid, a polymer-bound lipid (e.g., a pegylated lipid), and at least one structural lipid (e.g., a neutral lipid and a steroid or steroid analog). In some embodiments, 1,2, 3 or more of the aforementioned lipids may be excluded from the lipid nanoparticle.

In some embodiments, the lipid nanoparticle comprises a cationic lipid. In some embodiments, the cationic lipid is (4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (ALC-0315).

In some embodiments, the lipid nanoparticle comprises a polymer-bound lipid. In some embodiments, the lipid nanoparticle comprises a pegylated lipid, also known as a PEG-lipid. In some embodiments, the pegylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC or PEG-CerC), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamides, glycol-lipids including PEG-c-DOMG, PEG-c-DMA, PEG-S-DMG, N- [ (methoxypolyethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG, pegylated diacylglycerols (PEG-DAG) such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerols (PEG-DMG), pegylated phosphatidylethanolamine (PEG-PE), PEG succinic diacylglycerols (PEG-S-G) such as 4-O- (2 ',3' -di (tetradecyloxy) propyl-O-1-methoxy-ethyl) ceramide (PEG-co-propyl) or PEG-co-propyl-ethoxy) ceramide, such as co-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl-N- (ω -methoxy (polyethoxy) ethyl) carbamate. In some embodiments, 1,2,3, 4, 5 or more of the foregoing pegylated lipids may be excluded from the RNA molecule. In some embodiments, the pegylated lipid is 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159).

In some embodiments, the lipid nanoparticle comprises at least one structural lipid, such as a neutral lipid. In some embodiments, the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DPPC), ditalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl-oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dioleoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (sop), and/or 1, 2-dioleoyl-phosphatidylethanolamine (DOPE-PE). In some embodiments, 1,2, 3,4, 5 or more of the foregoing structural lipids may be excluded from the RNA molecules. In some embodiments, the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC).

In some embodiments, the lipid nanoparticle comprises a second structural lipid, such as a steroid or steroid analog. In some embodiments, the steroid or steroid analog is cholesterol.

In some embodiments, the lipid nanoparticle has an average diameter of about 1 to about 500nm, e.g., at least, up to, just below, or between any two of the following (inclusive or exclusive )：1nm、10nm、20nm、30nm、40nm、50nm、60nm、70nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm、150nm、160nm、170nm、180nm、190nm、200nm、210nm、220nm、230nm、240nm、250nm、260nm、270nm、280nm、290nm、300nm、310nm、320nm、330nm、340nm、350nm、360nm、370nm、380nm、390nm、400nm、410nm、420nm、430nm、440nm、450nm、460nm、470nm、480nm、490nm or 500nm.

In some embodiments, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising RNA molecules/polynucleotides encoding the RSV polypeptides disclosed herein in a concentration of at least, up to, just below, or between any two of (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01-0.09 mg/mL, encapsulated in an LNP comprising a lipid composition at or about 0.8-0.95 mg/mL (e.g., at least, At most, just below, or between any two of (inclusive or exclusive) 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL), a cationic lipid at or about 0.05 to 0.15mg/mL (e.g., at least, at most, just below, or between any two of (inclusive or exclusive) 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg/mL), a first structured lipid at or about 0.1 to 0.25mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive of) 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg/mL), and a first structured lipid at or about 0.3 to 0.45mg/mL (e.g., at least up to, just below, or between any two of (inclusive or exclusive) 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL). in some embodiments, the liquid composition further comprises a buffer composition comprising a first buffer at or between about 0.1-0.3 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive )：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29 or 0.30 mg/mL), at or between about 1.25-1.4 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive) 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL), and a stabilizer at or about 95-110 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive of) 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg/mL). in some embodiments, 1, 2, 3,4, 5 or more of the foregoing elements may be excluded from the liquid RNA-LNP composition. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing element concentrations may be excluded from the liquid RNA-LNP composition.

In particular embodiments, the liquid RNA-LNP immunogenic composition comprises RNA molecules/polynucleotides encoding the RSV polypeptides disclosed herein in a concentration of at least, up to, just below, or between any two of (including or exclusive to) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01-0.09 mg/mL, encapsulated in an LNP comprising a lipid composition at a concentration of at or about 0.8-0.95 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive to) 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95mg/mL ((4-hydroxybutyl) aminoxy) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315), at or about 0.05 to 0.15mg/mL (e.g., at least, up to, just below or between any two of the following (inclusive or exclusive) 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15mg/mL of 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159), at or about 0.1 to 0.25mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive of) 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22), 0.23, 0.24, or 0.25 mg/mL) of 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), and cholesterol at a concentration of or about 0.3-0.45 mg/mL (e.g., at least, up to, just below, or between any two of, including or exclusive of, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL). In some embodiments, the liquid composition further comprises a Tris buffer composition comprising bradykinin at a concentration of or about 0.1-0.3 mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive )：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29 or 0.30 mg/mL) and bradykinin at a concentration of or about 1.25-1.4 mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive) 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39 or 1.40 mg/mL) Tris hydrochloric acid (HCl), and sucrose at a concentration of or about 95-110 mg/mL (e.g., at least, up to, just below or between any two of (inclusive or exclusive of) 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 or 110 mg/mL). in some embodiments, 1, 2, 3,4, 5 or more of the foregoing elements may be excluded from the liquid RNA-LNP composition. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing element concentrations may be excluded from the liquid RNA-LNP composition.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises RNA molecules/polynucleotides encoding the RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between any two of (including or exclusive of): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90mg/mL, preferably or about 0.01-0.09 mg/mL, encapsulated in LNP and further comprising a Tris buffer at or about 5-15 mM (e.g., at least, up to, just below or between any two of (including or exclusivity) 5, 6, 7, 8, 9,10, 11, 12, 13, 14 or 15 mM) and sucrose at or about 200-400 mM (e.g., at least, up to, just below or between any two of (including or exclusivity) 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400 mM), the pH being at or about 7.0-8.0 (e.g., at least, up to, just below or between any two of (including or between) 7.1, 7.7, 7.0, 7.7.7, 7.7.7.0, 7.7.7.8, 7.7.7.0, 7.0, 7.0.0, or 7.0.0. In some embodiments, 1,2, 3, or more of the foregoing elements may be excluded from the liquid RNA-LNP composition. In some embodiments, 1,2, 3,4,5 or more of the foregoing element concentrations may be excluded from the liquid RNA-LNP composition.

In some embodiments, the RNA-LNP immunogenic composition is a lyophilized (reconstituted) RNA-LNP composition comprising RNA molecules/polynucleotides encoding RSV polypeptides disclosed herein in a concentration of at least, up to, just below, or between any two of (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01-0.09 mg/mL, encapsulated in an LNP comprising a lipid composition at a concentration of at or about 0.8-0.95 mg/mL (e.g., at least, At most, just below, or between any two of (inclusive or exclusive) 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL), a cationic lipid at or about 0.05 to 0.15mg/mL (e.g., at least, at most, just below, or between any two of (inclusive or exclusive) 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg/mL), a first structured lipid at or about 0.1 to 0.25mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive of) 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24 or 0.25 mg/mL), and a first structured lipid at or about 0.3 to 0.45mg/mL (e.g., at least up to, just below, or between any two of (inclusive or exclusive) 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL). In some embodiments, the lyophilized composition further comprises a first buffer at or about 0.01 and 0.15mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive) 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), a first buffer at or about 0.5 and 0.65mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive) 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL), a second buffer at or about 35 to 50mg/mL (e.g., at least, up to, just below, or between (including or exclusive of) any two of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL), a stabilizer at or about 5 to 15mg/mL (e.g., at least, just below, or between) for reconstitution, Up to, just below, or between any two of (inclusive or exclusive) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL). In particular embodiments, the lyophilized composition is reconstituted in or between about 0.6 to 0.75mL of a salt diluent (e.g., at least, up to, just below, or between any two of (including or exclusive of) 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75 mL). The concentration in the lyophilized RNA-LNP composition is determined after reconstitution. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing elements may be excluded from the lyophilized RNA-LNP composition. in some embodiments, 1,2, 3, 4, 5 or more of the foregoing element concentrations may be excluded from the lyophilized RNA-LNP composition.

In particular embodiments, the lyophilized (reconstituted) RNA-LNP composition comprises RNA polynucleotide encoding an RSV polypeptide disclosed herein in a concentration of at least, up to, just below, or between any two of (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01-0.09 mg/mL, encapsulated in LNP comprising a lipid composition at a concentration of at or about 0.8-0.95 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive of) 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94 or 0.95 mg/mL), ALC-0315 at a concentration of or about 0.05 to 0.15mg/mL (e.g., at least, up to, just below or between any two of (inclusive or exclusive of) 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 or 0.15 mg/mL), 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25mg/mL (e.g., at least, up to, just below, or between any two of (inclusive or exclusive) 0.1 to 0.25 mg/mL), and DSPC at a concentration of or about 0.3 to 0.45mg/mL (e.g., at least, up to, just below, or between any two of (inclusive or exclusive) 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45 mg/mL) and further comprises a Tris buffer composition comprising a concentration of or about 0.01 to 0.15mg/mL (e.g., at least, up to, just below or between (inclusive or exclusive) any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), and Tris HCl having a concentration of or about 0.5-0.65 mg/mL (e.g., at least, up to, just below, or between any two of (including or exclusive of) 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL), Sucrose at a concentration of or between about 35-50 mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive) 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 mg/mL), and sodium chloride (NaCl) diluent for reconstitution at a concentration of or between about 5-15 mg/mL (e.g., at least, up to, just below or between any two of (including or exclusive) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/mL). In particular embodiments, the lyophilized composition is reconstituted in or between about 0.6 to 0.75mL sodium chloride (e.g., at least, up to, just below, or between any two of (including or exclusive of) 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75 mL). The concentration in the lyophilized RNA-LNP composition is determined after reconstitution. In some embodiments, 1,2, 3, 4, 5 or more of the foregoing elements may be excluded from the lyophilized RNA-LNP composition. in some embodiments, 1,2, 3, 4, 5 or more of the foregoing element concentrations may be excluded from the lyophilized RNA-LNP composition.

The invention provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered to a subject at a dose of at least, up to, just below, or between any two of (inclusive or exclusive) 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or more of RSV RNA encapsulated in LNPs per administration. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing concentrations of RSV RNA encapsulated in LNP may be excluded.

The present invention provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered in a single dose. The invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice (e.g., on day 0 and at or about day 7, day 0 and at or about day 14, day 0 and at or about day 21, day 0 and at or about day 28, day 0 and at or about day 60, day 0 and at or about day 90, day 0 and at or about day 120, day 0 and at or about day 150, day 0 and at or about day 180, day 0 and at or about day 1, day 0 and at or about day 2, day 0 and at or about day 3, day 0 and at or about day 6, day 0 and at or about day 9, day 0 and at or about day 12, day 0 and at or about day 18, day 0 and at or about year 2, day 0 and at or about day 5, or day 0 and at or about 10). The invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice on day 0 and after or about 2 months. The invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice on day 0 and after or about 6 months. The invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fourteen, or more times. In some embodiments, periodic boosting at 1-5 year intervals may be required to maintain the protective level of the antibody. The invention further provides for the administration of at least one supplemental dose. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing administration regimens may be excluded.

The present invention provides a method of inducing an immune response to RSV in an individual comprising administering to the individual an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein. The invention further provides the use of an RNA molecule, RNA-LNP and/or immunogenic composition described herein for the manufacture of a medicament for inducing an immune response against RSV in an individual.

The present invention provides a method of inducing an immune response against RSV in an individual comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP, or immunogenic composition described herein comprising at least one open reading frame encoding an RSV polypeptide. The invention further provides for the use of an RNA molecule and/or RNA-LNP, or immunogenic composition described herein comprising at least one open reading frame encoding an RSV polypeptide, for the manufacture of a medicament for inducing an immune response against RSV in an individual.

The present invention provides a method of inducing an immune response to RSV in an individual comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP, or composition described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest. The invention further provides for the use of an RNA molecule and/or RNA-LNP, or composition described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest, for the manufacture of a medicament for inducing an immune response against RSV in an individual.

The present invention provides a method of preventing, treating and/or ameliorating an infection, disease or condition in an individual comprising administering to the individual an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein. The invention further provides the use of an RNA molecule, RNA-LNP and/or immunogenic composition as described herein for the manufacture of a medicament for preventing, treating and/or ameliorating an infection, disease or condition in an individual. In some embodiments, the infection, disease, or condition is associated with RSV. In some embodiments, the infection, disease or condition is an acute lower respiratory tract infection (acute lower respiratory infection; ALRI), including pneumonia and bronchitis. In some embodiments, the infection, disease, or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis.

The present invention provides a method of preventing, treating and/or ameliorating an infection, disease or condition in a subject comprising administering to the subject an effective amount of an RNA molecule and/or RNA-LNP, or immunogenic composition described herein comprising at least one open reading frame encoding an RSV polypeptide. The invention further provides for the use of an RNA molecule and/or RNA-LNP, or immunogenic composition comprising at least one open reading frame encoding an RSV polypeptide, as described herein, for the manufacture of a medicament for preventing, treating and/or ameliorating an infection, disease or condition in a subject. In some embodiments, the infection, disease, or condition is associated with RSV. In some embodiments, the infection, disease, or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis. In some embodiments, the infection, disease, or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis.

The invention further provides a method of preventing, treating and/or ameliorating an infection, disease or condition in an individual comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP, or immunogenic composition described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest. The invention further provides for the use of an RNA molecule and/or RNA-LNP, or immunogenic composition described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest, for the manufacture of a medicament for preventing, treating and/or ameliorating an infection, disease or condition in an individual. In some embodiments, the infection, disease, or condition is associated with a gene of interest.

In some embodiments, the individual is at least, at most, just below, or between (inclusive or exclusive) 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, or 12 months of age, or 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years old, or older. In some embodiments, the individual is, at least, at most, or about less than 1 year old, 1 year old or older, 5 years old or older, 10 years old or older, 20 years old or older, 30 years old or older, 40 years old or older, 50 years old or older, 60 years old or older, 70 years old or older. In some embodiments, the individual is at or about 50 years of age or older. In some embodiments, 1,2,3, 4,5 or more of the foregoing age groups are not administered an RNA molecule and/or an RNA-LNP.

In some embodiments, the individual has immunocompetence. In some embodiments, the individual is immunocompromised.

The invention provides a method or use as described herein wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered as a vaccine. The invention provides a method or use as described herein wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered by intradermal, intramuscular or intranasal injection.

It is contemplated that any of the embodiments discussed in this specification may be implemented with respect to any of the methods or compositions of the invention, and vice versa. Furthermore, the compositions of the present invention may be used to carry out the methods of the present invention.

Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in the language of the "use" claims, such as the use of any compound, composition, or agent discussed herein to achieve or perform the described therapeutic, diagnostic, or physiological purpose or effect. The use of one or more compositions may be employed based on any of the methods described herein.

Other objects, features and advantages of the present invention will become apparent from the following embodiments. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[ Brief description of the drawings ]

FIGS. 1A-1E show immunogenicity of modRNA-LNP formulations of RSV 847 in mice. Female BALB/c mice (10/group) were vaccinated intramuscularly with the RSV 847 construct in the form of bivalent protein subunit (RSV 847a+b) or in the form of a modRNA-LNP formulation of monovalent (RSV 847A) or bivalent (RSV 847a+b) at indicated doses on days 0 and 21. On day 35 (2 weeks post dose 2 (PD 2)) serum was collected for RSV neutralization analysis and spleen was collected for T cell analysis (ELISpot and intracellular cytokine staining ICS analysis). Fig. 1A and 1B show the results of neutralization analysis of RSV a and B, expressed as 50% neutralization titers (each symbol represents titers from individual animals. Bars represent Geometric Mean Titers (GMT)). FIG. 1C shows the results of an ELISPot assay that measures the number of RSV A+ B F specific cells secreting IFN-gamma and is expressed as Spot Forming Cells (SFC)/million cells. FIGS. 1D and 1E show ICS analysis results, which measure cells expressing RSVA+BF specific IFN-gamma, expressed as a percentage of IFN-gamma+ cells, within CD4 ⁺ and CD8 ⁺ T cells. Bars and error bars depict the median and quartile ranges. NA was not analyzed.

FIG. 2 shows immunogenicity of modRNA-LNP formulations encoding different RSV A pre-fusion F (preF) designs in mice. Female BALB/c mice (10/group) were vaccinated intramuscularly at a dose of 0.5 μg on days 0 and 21 with modRNA-LNP formulation encoding the pre-fusion F (preF) design of RSV a as described herein. On day 35 (2 w PD2), serum was analyzed for RSV a neutralization, expressed as 50% neutralization titer. Each symbol represents a titer from an individual animal. Bars represent Geometric Mean Titers (GMT).

FIGS. 3A-3F show immunogenicity of modRNA-LNP and sarA-LNP formulations of pre-RSV fusion F (preF) in mice. Female BALB/c mice (10/group) were vaccinated intramuscularly with RSV preF constructs in the form of bivalent protein subunits (RSV preF A +b) or bivalent modRNA-LNP formulations or bivalent saRNA-LNP formulations at indicated doses on days 0 and 21. Serum was collected for RSV neutralization analysis on day 21 (3 w PD1) and 35 (2 w PD2), and spleen was collected for T cell analysis on day 35 (intracellular cytokine staining ICS analysis). Neutralization assay results for RSV a and B are shown, expressed as 50% neutralization titers at 3w PD1 (fig. 3A and 3B) or 2w PD2 (fig. 3C and 3D). Each symbol represents a titer from an individual animal. Bars represent Geometric Mean Titers (GMT). FIGS. 3E and 3F show ICS analysis results, which measure cells expressing RSV preF A + B F specific IFN-. Gamma.within CD4 ⁺ T cells and CD8 ⁺ T cells. Bars and error bars depict the median and quartile ranges. NT, untested.

FIG. 4 schematically depicts wild-type (WT) RSV F protein (RSV WT) and variant RSV F protein constructs, wherein "SP" refers to the signal peptide sequence (amino acid residues 1-25 of each construct), "TM" refers to the transmembrane peptide sequence corresponding to the protein portion spanning the cell membrane, "CT" refers to the cytoplasmic tail peptide sequence corresponding to the protein portion extending into the cytoplasm of the cell, and "ectodomain" refers to the peptide sequence corresponding to the protein portion extending into the extracellular space, wherein the ectodomain comprises amino acid residues 1-513 (no TM and CT, denoted by "DeltaTM & CT"). Wherein the amino acid position of each moiety (i.e., SP, F2, pep27, F1) or mutation of each construct is indicated, e.g., SP of each construct spans amino acid residues 1-25 of each construct.

[ Embodiment ]

The present invention provides an RNA molecule (e.g., RNA polynucleotide) comprising at least one Open Reading Frame (ORF) encoding a Respiratory Syncytial Virus (RSV) antigen. In some embodiments, the RSV antigen is an RSV polypeptide. In some embodiments, the RSV polypeptide is an RSV F polypeptide. In some embodiments, the RSV polypeptides comprise an amino acid sequence set forth in table 1. In some embodiments, the RNA molecule comprises an ORF transcribed from at least one DNA nucleic acid sequence of table 2. In some embodiments, the RNA molecule comprises an ORF comprising the RNA nucleic acid sequence of table 3. In some embodiments, the RNA molecule comprises at least one of a 5' cap, a 5' utr, a 3' utr, and a poly a tail. In other embodiments, the RNA molecule comprises at least one of a5 'cap, a 3' utr, and a poly a tail. The present invention provides an RNA molecule comprising modified nucleotides (e.g., modified RNA; modRNA).

The present invention provides an immunogenic composition comprising any of the RNA molecules encoding the RSV polypeptides described herein complexed with, encapsulated in, or formulated with one or more lipids and formed into lipid nanoparticles (RNA-LNP). The invention further provides an immunogenic composition comprising any of the RNA molecules comprising at least one RNA nucleic acid described herein complexed with, encapsulated in, or formulated with one or more lipids and forming RNA-LNP. The invention further provides a method of preventing, treating, or ameliorating an infection, disease, or condition (e.g., an RSV infection-associated respiratory disorder, including pneumonia and bronchitis) in a subject via administering to the subject an effective amount of an RNA molecule, RNA-LNP, or immunogenic composition described herein. The invention further provides the use of an RNA molecule, RNA-LNP and/or immunogenic composition as described herein as a vaccine.

The invention may be understood more readily by reference to the following detailed description of embodiments of the invention and the embodiments included therein. It is to be understood that the present invention is not limited to a particular method of manufacture, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

All references cited herein, including patent applications, patent publications, uniProtKB accession numbers, are incorporated herein by reference as if each individual reference were specifically and individually indicated to be incorporated by reference in its entirety.

[ I ] defined examples ]

Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art.

Throughout this disclosure, the terms "about" and "approximately" and "substantially" are used in accordance with their ordinary and customary meaning in the art of cell and molecular biology to indicate a deviation of + -10% of one or more values associated therewith. Thus, in any disclosed embodiment, the term may be replaced by "within a [ percent ] of" specified content ". In one non-limiting embodiment, the percentage includes 0.1%, 0.5%, 1%, 5% and 10%.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

The use of the word "a" or "an" when used in conjunction with the term "comprising" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

The phrase "and/or" means "or". For example A, B and/or C include A alone, B alone, C, A alone in combination with B, A in combination with C, B in combination with C, or A, B and C. In other words, "and/or" is used as an inclusion or operation.

The phrase "substantially all" is defined as "at least 95%", and if substantially all members of a group have a certain characteristic, at least 95% of the members of the group have that characteristic. In some embodiments, substantially all means that a group member equal to, at least any of, or between any of the following has the characteristic 95%, 96%, 97%, 98%, 99%, or 100%.

The compositions and methods of use thereof may "comprise," consist essentially of, "or" consist of any of the ingredients or steps disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, the words "comprise" (and any form of comprising such as "comprises" and "comprising)", "having" (and any form of having such as "having" and "having)", "including" (and any form of comprising such as "including" and "including)") or "containing" (and any form of comprising such as "including" and "including") are inclusive or open-ended, and are to be understood as implying a group comprising the stated step or element or step or element, but not excluding any other step or element or group of steps or elements. It is contemplated that embodiments described herein in the context of the term "comprising" may also be practiced in the context of the term "consisting of or" consisting essentially of. Compositions and methods that "consist essentially of" any of the disclosed components or steps limit the scope of the claims to specific materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. The word "consisting of (consisting of)" (and any form of consisting of (consist of) ", such as" consisting of (consists of) ") is meant to include and be limited to anything following the phrase" consisting of (consists of) ". Thus, the phrase "consisting of" indicates that the listed elements are required or necessary, and that no other elements may be present.

Reference throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The term "inhibit (inhibiting)", "reduce (decreasing)", or "reduce (reducing)", or any variation of these terms, includes any measurable reduction (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction) or complete inhibition to achieve the desired result. The term "improvement", "promoting (promote)" or "increasing (increase)" or any variant of these terms includes any measurable increase (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% increase) to achieve a desired result or yield of a protein or molecule.

As used herein, the terms "reference," "standard," or "control" describe a value relative to which a comparison is made. For example, an agent, individual, population, sample, or value of interest is compared to a reference, standard, or control agent, individual, population, sample, or value of interest. The reference, standard or control may be tested and/or assayed substantially simultaneously and/or together with a test or assay of interest of an agent, individual, population, sample or value of interest and/or may be assayed or characterized under conditions or circumstances commensurate with the agent, individual, population, sample or value of interest under evaluation.

The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or culture medium from which it originates (when produced by recombinant DNA technology), or chemical precursors or other chemicals (when chemically synthesized). Further, an isolated compound refers to a compound that can be administered to an individual as an isolated compound, in other words, if the compound adheres to a column or is embedded in an agarose gel, it cannot be simply considered "isolated". Furthermore, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally exist in the form of a fragment and/or is not normally in a functional state and/or is altered or removed from the natural state via manual intervention. For example, DNA naturally present in living animals is not "isolated," but synthetic DNA or DNA partially or completely isolated from coexisting materials in its natural state is "isolated. The isolated nucleic acid may be present in a substantially purified form, or may be present in a non-natural environment, such as a cell into which the nucleic acid has been delivered.

As used herein, "nucleic acid" is a molecule comprising a nucleic acid component and refers to a DNA or RNA molecule. It is used interchangeably with the term "polynucleotide". A nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers covalently linked to each other by phosphodiester linkages of a sugar/phosphate backbone. Nucleic acids may also encompass modified nucleic acid molecules, such as DNA or RNA molecules with base modifications, sugar modifications, or backbone modifications. The nucleic acid may be present in a variety of forms, such as an isolated segment of incorporated sequence and a recombinant vector or recombinant polynucleotide encoding a polypeptide such as an antigen or antibody, or a fragment, derivative, mutein or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides, antisense nucleic acids for inhibiting expression of the polynucleotides, mRNA, saRNA, modRNA and complementary sequences described herein before. The nucleic acid may encode an epitope to which the antibody may bind.

The term "epitope" refers to a moiety specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope comprises a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts a related three-dimensional configuration. In some embodiments, when the antigen adopts such a configuration, such chemical atoms or groups are physically close to each other in space. In some embodiments, when the antigen adopts an alternative configuration (e.g., is linearized), at least some such chemical atoms or groups are physically separated from each other.

The nucleic acid may be single-stranded or double-stranded, and may comprise RNA and/or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids). In some cases, the nucleic acid sequence may encode a polypeptide sequence with other heterologous coding sequences, e.g., to achieve purification, transport, secretion, post-translational modification of the polypeptide, or to achieve a therapeutic benefit, such as targeting or efficacy. A tag or other heterologous polypeptide may be added to the modified polypeptide coding sequence, wherein "heterologous" refers to a polypeptide that is different from the modified polypeptide.

The term "polynucleotide" refers to a nucleic acid molecule that may be recombinant or that has been isolated from total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors (including, for example, plasmids, cosmids, phages, viruses), and the like. In certain embodiments, the polynucleotide comprises regulatory sequences substantially separate from the naturally occurring gene or protein coding sequence thereof. The polynucleotide may be single-stranded (encoding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may (but need not) be present within the polynucleotide.

In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein, those comprising sequence identity equal to, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more than any two of any one of, at least any one of, at most any one of, or any two of the following compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain embodiments, an isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90% identity to an amino acid sequence described herein over the entire sequence length, or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, an isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 95% identity to an amino acid sequence described herein over the entire sequence length, or a nucleotide sequence complementary to the isolated polynucleotide.

Regardless of the length of the coding sequence itself, the nucleic acid segment may be combined with other nucleic acid sequences (such as promoters, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, etc.), such that the overall length thereof may vary significantly. The nucleic acid may have any length. The nucleic acid can be, for example, equal to any one of the following, at least any one of the following, at most any one of the following, or between any two of the following ：5、10、15、20、25、30、35、40、45、50、75、100、125、175、200、250、300、350、400、450、500、750、1000、1500、3000、5000、6000、7000、8000、9000、10000、11000、12000、13000、14000、15000 or more nucleotides in length, and/or can comprise one or more additional sequences (e.g., regulatory sequences), and/or can be part of a larger nucleic acid (e.g., vector). It is therefore contemplated that almost any length of nucleic acid fragment may be used, with the overall length being limited by ease of preparation and intended use in recombinant nucleic acid protocols.

In this regard, the term "gene" is used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those of skill in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or a portion of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid comprising a variation that has a slightly different nucleic acid sequence, but still encodes the same or substantially similar polypeptide.

As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product by the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of the nucleic acid sequence involves one or more of (1) generating an RNA template from the DNA sequence (e.g., by transcription), (2) processing the RNA transcript (e.g., by splicing, editing, etc.), (3) translating the RNA into a polypeptide or protein, and/or (4) post-translationally modifying the polypeptide or protein.

In general, the term "engineered" refers to an embodiment that has been manually manipulated. For example, a polynucleotide is considered "engineered" when two or more sequences that are not linked together in order in nature are manually manipulated to be directly linked to each other in an engineered polynucleotide, and/or when particular residues in the polynucleotide are non-naturally occurring and/or are caused to be linked to entities or portions thereof that are not linked in nature via the action of a human.

As used herein, the term "DNA" means a nucleic acid molecule comprising nucleotides such as deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate monomers, consisting of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and polymerized from a characteristic backbone structure. The backbone structure is typically formed by phosphodiester bonds between the sugar moiety (e.g., deoxyribose) of a nucleotide of a first adjacent monomer and the phosphate moiety of a second adjacent monomer. The specific order of monomers (e.g., the order of bases attached to the sugar/phosphate backbone) is referred to as the DNA sequence. The DNA may be single-stranded or double-stranded. In double-stranded form, the nucleotides of the first strand typically hybridize to the nucleotides of the second strand, e.g., by A/T base pairing and G/C base pairing. The DNA may comprise all or most deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide lacking a hydroxyl group at the 2' position of the β -D-ribofuranosyl group. Without any limitation, DNA may encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

As used herein, the term "RNA" means a nucleic acid molecule comprising nucleotides such as adenosine monophosphate, uridine monophosphate, guanosine monophosphate, and cytidine monophosphate monomers, which are linked to each other along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar (e.g., ribose) of a first monomer and the phosphate moiety of a second, adjacent monomer. RNA can be obtained, for example, in cells by transcription of DNA sequences. In eukaryotic cells, transcription is usually performed in the nucleus or in the granulosa line. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). For example, processing of immature RNA in eukaryotic organisms involves various post-transcriptional modifications such as splicing, 5' capping, polyadenylation, export from the nucleus or centrosome. Processing mature messenger RNA and providing a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. Mature mRNA can comprise a 5' cap, 5' UTR, open reading frame, 3' UTR, and polyadenylation tail sequence. The RNA may comprise all or most ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide that has a hydroxy group at the 2' position of the β -D-ribofuranosyl group. In one embodiment, the RNA can be messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known to those skilled in the art, mRNA will typically contain a 5 'untranslated region (5' UTR), a polypeptide coding region, and a 3 'untranslated region (3' UTR). Without any limitation, RNA can encompass double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA (modRNA).

An "isolated RNA" is defined as an RNA molecule that may be recombinant or that has been isolated from total genomic nucleic acid. The isolated RNA molecule or protein may be present in a substantially purified form, or may be present in a non-natural environment (such as a host cell).

"Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution, and/or alteration of one or more nucleotides as compared to a naturally occurring RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides, or to one or more 5 'and/or 3' ends of RNA. In one embodiment, such modRNA contains at least one modified nucleotide, such as a change in the base of a nucleotide. For example, the modified nucleotide may replace one or more uridine and/or cytidine nucleotides. For example, these substitutions may be made for each instance of uridine and/or cytidine in the RNA sequence, or may be made for only selected uridine and/or cytidine nucleotides. Such changes to standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide in the RNA sequence may be replaced with N1-methyl pseudouridine. Other such altered nucleotides are known to those skilled in the art. Such altered RNA molecules are considered analogues of naturally occurring RNAs. In some embodiments, RNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some embodiments, the RNA may be a replicon RNA (replicon), in particular a self-replicating RNA or a self-amplifying RNA (saRNA).

As contemplated herein, without limitation, RNA can be used as a therapeutic modality for treating and/or preventing a variety of conditions in mammals, including humans. The methods described herein comprise administering an RNA described herein to a mammal (such as a human). For example, in one embodiment, such methods of using RNA include RNA vaccines encoding antigens to induce stable neutralizing antibodies and accompanying T cell responses to achieve protective immunization. In some embodiments, a minimum vaccine dose is administered to induce robust neutralizing antibodies and concomitant T cell responses to achieve protective immunization. In one embodiment, the RNA administered is in vitro transcribed RNA. For example, such RNAs can be used to encode at least one antigen intended to generate an immune response in the mammal. The pathogenic antigen is a peptide or protein antigen derived from a pathogen associated with an infectious disease. In certain embodiments, the pathogenic agent is a peptide or protein antigen derived from RSV. Conditions and/or diseases treatable with the RNAs disclosed herein include, but are not limited to, those caused by and/or affected by viral infection. Such viruses include, but are not limited to, RSV.

As used herein, "prevent (Prevent)" or "prevention" when used in connection with the occurrence of a disease, disorder, and/or condition refers to reducing the risk of suffering from a disease, disorder, and/or condition and/or delaying the onset of one or more features or symptoms of the disease, disorder, or condition. Prevention may be considered complete when the onset of the disease, disorder or condition has been delayed for a predetermined period of time.

As will be appreciated from the context, "risk" of a disease, disorder, and/or condition refers to the likelihood that a particular individual will suffer from the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, the risk is, at least, or at most 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, the risk is expressed as a risk relative to a risk associated with a reference sample or a group of reference samples. In some embodiments, the reference sample or group of reference samples has a known risk of a disease, disorder, condition, and/or event. In some embodiments, the reference sample or group of reference samples is from an individual similar to the particular individual. In some embodiments, the risk may reflect one or more genetic attributes, e.g., which may predispose an individual to (or not) a particular disease, disorder, and/or condition. In some embodiments, the risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. An individual who is "susceptible" to a disease, disorder, and/or condition is an individual who is at a higher risk of suffering from the disease, disorder, and/or condition than a member of the general public. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will suffer from the disease, disorder, and/or condition. In some embodiments, an individual susceptible to a disease, disorder, and/or condition will not suffer from the disease, disorder, and/or condition.

The terms "protein", "polypeptide" or "peptide" are used synonymously herein and refer to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, xenogeneic homologs, fragments, and other equivalents, variants, and analogs of the foregoing. The polypeptide may be a single molecule or may be a multi-molecular complex, such as a dimer, trimer or tetramer. Proteins comprise one or more peptides or polypeptides and may be folded into a 3-dimensional form that may be required for the protein to perform its biological function.

As used herein, the term "wild-type" or "WT" or "native" refers to an endogenous form of a molecule that naturally occurs in an organism. In some embodiments, wild-type forms of the protein or polypeptide are employed, however in other embodiments of the invention, modified proteins or polypeptides are used to generate an immune response. The terms described above are used interchangeably.

"Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, and in particular its amino acid sequence, is altered relative to the wild-type protein or polypeptide. In some embodiments, the modified protein/variant protein or polypeptide has at least one modified activity or function (recognizing that the protein or polypeptide may have a variety of activities or functions). In particular, it is contemplated that the modified/variant protein or polypeptide may be altered relative to one activity or function, but in other embodiments retains wild-type activity or function, such as immunogenicity. When referring specifically herein to a protein, it generally refers to either a natural (wild-type) or recombinant (modified) protein. Proteins can be isolated directly from the native organism, produced by recombinant DNA/exogenous expression methods or produced by solid phase peptide synthesis (solid-PHASE PEPTIDE SYNTHESIS; SPPS) or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence encoding a polypeptide (e.g., an antigen or fragment thereof). The term "recombinant" may be used in conjunction with a polypeptide or the name of a particular polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or a polypeptide that is a replication product of such a molecule.

The term "fragment" of a reference amino acid sequence (peptide or protein) refers to a portion of an amino acid sequence, i.e. a sequence representing an amino acid sequence shortened at the N-and/or C-terminus. The fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' -end of the open reading frame. The shortened fragment at the N-terminus (C-terminal fragment) may be obtained, for example, by translating a truncated open reading frame at the 5' -end lacking the open reading frame, provided that the truncated open reading frame comprises an initiation codon for initiating translation. Fragments of an amino acid sequence comprise, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 99% of the amino acid residues from the amino acid sequence. In the present invention, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having at least, up to, just below, or any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived.

In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 70% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 80% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 85% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 90% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 95% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 97% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA nucleic acid, or RNA nucleic acid sequence refers to a sequence having at least 99% sequence identity to the polypeptide, DNA nucleic acid, or RNA nucleic acid sequence from which it is derived.

As used herein in the context of a molecule (e.g., a nucleic acid, protein, or small molecule), the term "variant" refers to a molecule that exhibits significant structural identity to a reference molecule but is structurally different from the reference molecule, e.g., in the presence or absence of one or more chemical moieties or in the content of one or more chemical moieties as compared to a reference entity. In some embodiments, the variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity to the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural components. by definition, a variant is a different molecule that shares one or more such feature modules with a reference molecule, but differs from the reference molecule in at least one embodiment. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid due to one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrate, lipid, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., linked to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid exhibits at least, up to, just below, or any two of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence component with a reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduction in one or more levels of biological activity as compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if its amino acid or nucleotide sequence is identical to that of the reference polypeptide or nucleic acid but has a few sequence changes at a particular position. Preferably, the variant polypeptide or nucleic acid sequence has at least one modification, e.g., 1 to about 20 modifications, as compared to a reference polypeptide or nucleic acid sequence. In one embodiment, the variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, the variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, the variant polypeptide or nucleic acid sequence has 1 to about 4 modifications compared to a reference polypeptide or nucleic acid sequence. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted as compared to the reference. Typically, a variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (e.g., residues involved in a particular biological activity) relative to a reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue as compared to a reference. In some embodiments, a variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions as compared to a reference. In some embodiments, a variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions as compared to a reference, and in some embodiments, no additions or deletions.

In some embodiments, the reference polypeptide or nucleic acid is a "wild-type" or "WT" or "native" sequence found in nature, including allelic variation. The wild-type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein or polypeptide) comprises an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and/or an amino acid substitution variant. "variants" of a nucleotide sequence include nucleotide insertion variants, nucleotide addition variants, nucleotide deletion variants and/or nucleotide substitution variants. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants and species homologs, in particular naturally occurring ones of the above. The term "variant" includes in particular fragments of amino acid or nucleic acid sequences.

Changes may be introduced into a nucleic acid by mutation, thereby causing an alteration in the amino acid sequence of a polypeptide (e.g., antigen or antibody derivative) encoded by the nucleic acid. Mutations may be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered using, for example, a random mutation-inducing scheme. In some embodiments, regardless of the manner in which it is performed, mutant polypeptides may be expressed and screened for desired characteristics.

Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions may be made that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations that selectively alter the biological activity of a polypeptide encoded by a nucleic acid may be introduced into the nucleic acid. For example, mutations can alter biological activity quantitatively or qualitatively. Examples of quantitative alterations include increasing, decreasing or eliminating activity. Examples of qualitative alterations include altering the antigen specificity of an antibody.

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

The terms "% identity"%, "identity"% identity "or similar terms are intended to refer in particular to the percentage of identical nucleotides or amino acids in the optimal alignment between the sequences to be compared. This percentage is purely statistical and the differences between the two sequences may, but need not, be randomly distributed over the length of the sequences being compared. The comparison of two sequences is typically performed by comparing the sequences after optimal alignment with respect to a comparison fragment or "window" in order to identify the local region of the corresponding sequence. The optimal alignment for comparison can be performed manually or by means of the local homology algorithm of Smith and Waterman,1981,Ads App.Math.2,482, by means of the local homology algorithm of NEDDLEMAN and Wunsch,1970, J.mol.biol.48,443, by means of the similarity search algorithm of Pearson and Lipman,1988,Proc.Natl Acad.Sci.USA 88,2444, or by means of a computer program using said algorithm (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA, genetics Computer Group in the Wisconsin genetics software package). In some embodiments, the percent identity of two sequences is determined using BLASTN or BLASTP algorithm available at United States National Center for Biotechnology Information (NCBI) website.

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

In some embodiments, the degree of similarity or identity is given for a region at least, up to, just below, or between any two of the reference sequences of about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given to at least, up to, just below, or between any two of the following (in some embodiments, consecutive nucleotides) about 100, about 120, about 140, about 160, about 180, or about 200. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

Homologous amino acid sequences may exhibit at least, up to, just below, or between any two of the following, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% identity of amino acid residues. In one embodiment, the homologous amino acid sequences exhibit at least 95% identity of the amino acid residues. In one embodiment, the homologous amino acid sequences exhibit at least 98% identity of the amino acid residues. In one embodiment, the homologous amino acid sequences exhibit at least 99% identity of the amino acid residues.

Fragments or variants of an amino acid sequence (peptide or protein) may be "functional fragments" or "functional variants". The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to the amino acid sequence from which it is derived, e.g., that is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence comprising an amino acid sequence that has one or more amino acid changes as compared to a parent molecule or sequence and that is still capable of performing one or more functions of the parent molecule or sequence, e.g., inducing an immune response. In one embodiment, modifications in the amino acid sequence of a parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. The term "mutant" of wild-type RSV F protein, "mutant" of RSV F protein, "RSV F protein mutant" or "modified RSV F protein" refers to a polypeptide that exhibits an introduced mutation relative to wild-type F protein and is immunogenic against wild-type F protein.

An amino acid sequence (peptide, protein or polypeptide) that is "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the particular sequence or fragment thereof. The amino acid sequence derived from a particular amino acid sequence may be a variant of that particular sequence or fragment thereof. For example, one of ordinary skill in the art will appreciate that an antigen suitable for use herein may be altered such that it differs in sequence from the naturally occurring sequence or native sequence from which it is derived, while retaining the desired activity of the native sequence.

In the present invention, a vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. Vectors may be used to incorporate nucleic acid sequences, such as nucleic acid sequences comprising an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, transfer vectors. The vector may be an RNA vector or a DNA vector. In some embodiments, the vector is a DNA molecule. In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector. Typically, the expression vector will contain the desired coding sequence, or other sequences as appropriate necessary for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify a certain desired fragment (typically a DNA fragment), and may lack the functional sequences required to express one or more desired fragments.

As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be an immunogenic composition. In some embodiments, the active agent is present in the treatment regimen in an amount that is appropriate for the unit dose administered, which exhibits a statistically significant probability of achieving the intended therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition may be formulated specifically for parenteral administration, e.g., in the form of a sterile solution or suspension, or a sustained release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection.

As used herein, the term "vaccination" refers to the administration of an immunogenic composition intended to produce an immune response, such as a disease-related (e.g., pathogenic) agent (e.g., a virus). In some embodiments, vaccination may be administered before, during, and/or after exposure to a disease-related agent, and in some embodiments, immediately before, during, and/or after exposure to the agent. In some embodiments, vaccination comprises multiple administrations of the vaccine composition appropriately spaced apart in time. In some embodiments, vaccination generates an immune response against an infectious agent. In some embodiments, vaccination generates an immune response against a tumor, in some such embodiments, vaccination is "personalized" in that it is directed partially or fully against one or more epitopes (e.g., which may be or include one or more neoepitopes) determined to be present in a tumor of a particular individual.

Immune response refers to a humoral response, a cellular response, or both a humoral response and a cellular response in an organism. The immune response may be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize proteins or cell surface proteins, assays that measure T cell activation or proliferation, and/or assays that measure modulation of the activity or expression of one or more cytokines.

As used herein, the term "combination therapy" refers to those instances in which an individual is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens may be administered simultaneously, in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered prior to any doses of the second regimen), in some embodiments, such agents are administered in overlapping administration regimens. In some embodiments, "administering" of a combination therapy may involve administering one or more agents or one or more modalities to an individual receiving one or more other agents or one or more modalities in combination (modality). For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even must be at the same time), but in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Those skilled in the art will appreciate that the term "administration regimen" may be used to refer to a set of unit doses (typically more than one) administered individually to an individual, typically at intervals of time. In some embodiments, a given therapeutic agent has a recommended administration regimen, which may involve one or more doses. In some embodiments, the administration regimen comprises a plurality of doses, each of the doses being separated in time from the other doses. In some embodiments, the individual doses are spaced apart from each other by a period of equal length, and in some embodiments, the administration regimen comprises a plurality of doses and at least two different periods separating the individual doses. In some embodiments, all doses within an administration regimen have the same amount of unit dose. In some embodiments, different doses within an administration regimen have different amounts. In some embodiments, the administration regimen comprises a first dose of an amount of the first dose followed by one or more additional doses of an amount of a second dose different from the amount of the first dose. In some embodiments, the administration regimen comprises a first dose in an amount of the first dose followed by one or more additional doses in an amount of the second dose that is the same as the amount of the first dose. In some embodiments, the administration regimen is associated with a desired or beneficial outcome (e.g., is a therapeutic administration regimen) when administered across the relevant population.

[ II ] Respiratory Syncytial Virus (RSV) ]

The present invention provides RNA molecules (e.g., RNA polynucleotides) comprising at least one open reading frame encoding a Respiratory Syncytial Virus (RSV) polypeptide. The invention further provides an immunogenic composition comprising at least one RNA molecule encoding an RSV polypeptide complexed with, encapsulated in, or formulated with one or more lipids and forming a Lipid Nanoparticle (LNP). The RSV polypeptides included in the immunogenic compositions disclosed herein can be any RSV F protein in a pre-fusion configuration.

The term "pre-fusion conformation" refers to the structural conformation adopted by either (i) the RSV F protein or mutant, when in monomeric or trimeric form, to be specifically bound by antibody D25 or AM22, or (ii) the RSV F protein or mutant thereof, when the RSV F protein mutant is in trimeric form, to be specifically bound by antibody AM 14. The pre-fusion trimer configurations are a subset of the pre-fusion configurations. As used herein, a RSV F protein or polypeptide or mutant thereof in a pre-fusion configuration can be expressed as "RSV preF".

The term "post-fusion conformation" refers to the structural conformation adopted by the RSV F protein that is not specifically bound by D25, AM22 or AM 14. The native F protein adopts a post-fusion configuration after fusion of the viral envelope with the host cell membrane. The RSV F protein can also take on a post-fusion conformation outside of the context of a fusion event, e.g., under stress conditions such as heat and low osmotic pressure, when extracted from the membrane, when expressed as an extracellular domain, or after storage. The term "AM14" refers to the antibody described in WO 2008/147196A2 (which is incorporated herein by reference in its entirety). The term "AM22" refers to an antibody described in WO 2011/043643 A1 (which is incorporated herein by reference in its entirety). The term "D25" refers to the antibody described in WO 2008/147196A2 (which is incorporated herein by reference in its entirety).

In some embodiments, the RSV F protein is a RSV F protein of subtype a. In some embodiments, the RSV F protein is an RSV F protein of subtype B. As used herein, the term "subtype" and "subgroup" are used interchangeably. As used herein, the term "strain" refers to a particular isolate within each subtype or subgroup. In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein. In some embodiments, the RSV F protein is a mutant of the wild-type RSV F protein of subtype a. In some embodiments, the RSV F protein is a mutant of the wild-type RSV F protein of subtype B. In some embodiments, the mutant exhibits an introduced mutation in the amino acid sequence relative to the amino acid sequence of a corresponding wild-type RSV F protein and is immunogenic against the wild-type RSV F protein in a pre-fusion configuration or against a virus comprising the wild-type F protein. Amino acid mutations in mutants include amino acid substitutions, deletions or additions relative to the wild-type RSV F protein.

In some embodiments, the RSV F protein is a RSV protein mutant described in WO2017/109629 (which is incorporated herein by reference in its entirety).

In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein, wherein the introduced amino acid mutation is a mutation of a pair of amino acid residues in the wild-type RSV F protein to a pair of cysteines ("engineered disulfide bond mutation"). The introduced pair of cysteine residues allows disulfide bond formation between the cysteine residues, which stabilizes the conformation or oligomeric state of the protein (such as the pre-fusion conformation). Examples of specific such pairs of mutations include 55C and 188C, 155C and 290C, 103C and 148C, and 142C and 371C, such as S55C and L188C, S155C and S290C, A103C and I148C, and L142C and N371C.

In still other embodiments, the RSV F protein mutants comprise an amino acid mutation that is one or more cavity filling mutations. Examples of target substituted amino acids that may be filled with cavities include small aliphatic (e.g., gly, ala, and Val) or small polar amino acids (e.g., ser and Thr), as well as amino acids that are buried in the pre-fusion configuration but exposed to solvent in the post-fusion configuration. Examples of substituted amino acids include larger aliphatic amino acids (Ile, leu, and Met) or larger aromatic amino acids (His, phe, tyr and Trp). In some specific embodiments, the RSV F protein mutant comprises a cavity filling mutation selected from the group consisting of:

(1) S at positions 55, 62, 155, 190 or 290 is substituted with I, Y, L, H or M;

(2) T at positions 54, 58, 189, 219 or 397 is substituted with I, Y, L, H or M;

(3) G at position 151 is substituted with A or H;

(4) A at position 147 or 298 is substituted with I, L, H or M;

(5) V at position 164, 187, 192, 207, 220, 296, 300 or 495 is substituted with I, Y, H, and

(6) R at position 106 is substituted with W.

In some particular embodiments, the RSV F protein mutants comprise at least one cavity filling mutation selected from the group consisting of T54H, S I and V296I.

In yet other embodiments, the RSV F protein mutants comprise electrostatic mutations that reduce ion repulsion between residues in proteins that are in close proximity to each other in the folded structure or increase ion attraction therebetween. In several embodiments, the RSV F protein mutants include electrostatic substitutions that reduce repulsive ionic interactions with or increase attractive ionic interactions with the acidic residues of Glu487 and Asp489 from another protomer of the RSV F trimer. In some particular embodiments, the RSV F protein mutant comprises an electrostatic mutation selected from the group consisting of:

(1) E at position 82, 92 or 487 is substituted with D, F, Q, T, S, L or H;

(2) K at position 315, 394 or 399 is substituted with F, M, R, S, L, I, Q or T;

(3) D in position 392, 486 or 489 is substituted with H, S, N, T or P, and

(4) R at positions 106 or 339 is substituted with F, Q, N or W.

In still other embodiments, the RSV F protein mutant comprises a combination of two or more different types of mutations selected from the group consisting of engineered disulfide mutations, cavity filling mutations and electrostatic mutations. In some particular embodiments, the RSV F protein mutants comprise a combination of mutations relative to the corresponding wild-type RSV F protein, wherein the combination of mutations is selected from the group consisting of:

(1) A103C, I, 148, C, S, 190I, D486S;

(2) A combination of T54H, S, 55C, L, C, D486S;

(3) A combination of T54H, A103C, I148C, S190I, V296I and D486S;

(4) A combination of T54H, S, 55C, L, 142C, L, 188C, V296I and N371C;

(5) S55C, L188C and D486S;

(6) A combination of T54H, S, 55, C, L, 188C and S190I;

(7) A combination of S55C, L188,188, 188C, S190,190I and D486S;

(8) A combination of T54H, S, 55C, L, 188, C, S, 190I and D486S;

(9) S155C, S, 190, I, S, 290C and D486S;

(10) A combination of T54H, S, 55C, L, 142C, L, 188C, V, 296I, N371C, D486S, E487Q and D489S;

(11) T54H, S155C, S190I, S290C and V296I, and

(12) S155C, S, 190, F, S, 290C and V207L.

In some embodiments, the RSV F protein has subtype a and includes mutations S155C, S, 190F, S, 290C and V207L.

In some embodiments, the RSV F protein has subtype B and includes mutations S155C, S, 190, F, S, 290C and V207L.

In some embodiments, the RSV F protein has subtype a and comprises mutations S155C, S F and S290C.

In some embodiments, the RSV F protein has subtype B and comprises mutations S155C, S F and S290C.

In some embodiments, the RSV F protein has subtype a and includes mutations a103C, I148C, S I and D486S.

In some embodiments, the RSV F protein has subtype B and includes mutations a103C, I148C, S I and D486S.

In some embodiments, the RSV F protein has subtype a and includes mutations T54H, A103C, I148C, S I and D486S.

In some embodiments, the RSV F protein has subtype B and includes mutations T54H, A103C, I148C, S I and D486S.

In some embodiments, the RSV F protein has subtype a and includes mutations T54H, S, C, L188C and D486S.

In some embodiments, the RSV F protein has subtype B and includes mutations T54H, S, C, L188C and D486S.

Given the substantial conservation of RSV F sequences, one of ordinary skill in the art can readily compare amino acid positions between different natural RSV F sequences to identify corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, the furin cleavage site falls within the same amino acid position throughout almost all of the identified natural RSV F0 precursor proteins. Thus, conservation of the native RSV F protein sequence throughout the strain and subtype allows the use of reference RSV F sequences for comparison of amino acids at specific positions in the RSV F protein. For the purposes of the present invention (unless the context indicates otherwise), the amino acid positions of the RSV F protein are given with reference to the amino acid sequence of the full length natural F precursor polypeptide of the RSV A2 strain, corresponding to GenInfo identifier GI 138251 and Swiss Prot identifier P03420 (SEQ ID NO: 1).

In some embodiments, the RSV F protein is a mature form of the RSV F protein that comprises two individual polypeptide chains, an F1 polypeptide and an F2 polypeptide. In some other embodiments, the F2 polypeptide is linked to the F1 polypeptide by one or two disulfide bonds to form an F2/F1 heterodimer. In still other embodiments, the RSV F mutant is in the form of a single chain protein, wherein the F2 polypeptide is linked to the F1 polypeptide by a peptide bond or a peptide linker. Any suitable peptide linker for joining two polypeptide chains together may be used. Examples of such linkers include G, GG, GGG, GS and SAIG linker sequences. The linker may also be a full length pep27 sequence or a fragment thereof, which full length pep27 sequence corresponds to amino acids at positions 110 to 136 of SEQ ID NO. 1.

The mutant F1 polypeptide chain may have the same length as the full-length F1 polypeptide corresponding to the wild-type RSV F protein, however, it may also have a deletion, such as a deletion of 1-60 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full length F1 polypeptide of the RSV F mutant corresponds to amino acid positions 137-574 of the natural RSV F0 precursor (SEQ ID NO: 1) and comprises (from N-terminus to C-terminus) an extracellular region (residues 137-524), a transmembrane domain ("TM") (residues 525-550) and a cytoplasmic domain ("CT") (residues 551-574). It should be noted that amino acid residue 514 from the native F1 polypeptide sequence is an optionally present sequence in the F1 polypeptide of the RSV F protein included in the immunogenic compositions provided herein, and thus may not be present in the F1 polypeptide of the mutant.

In some embodiments, the F1 polypeptide of the RSV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks a cytoplasmic domain as well as a portion or all of a transmembrane domain. In some particular embodiments, the mutant comprises an F1 polypeptide in which the amino acid residue from position 510, 511, 512, 513, 514, 515, 520, 525, or 530-574 is absent. Typically, amino acids 514-574 may not be present for mutants linked to trimerization domains, such as foldon. Thus, in some particular embodiments, amino acid residues 514-574 are not present in the mutant F1 polypeptide. In yet other specific embodiments, the F1 polypeptide of the RSV F mutant comprises or consists of amino acid residues 137-513 of the native F0 polypeptide sequence (SEQ ID NO: 1), such as the RSV 847A-fold sub-polypeptide (SEQ ID NO: 74), or alternatively any of the F0 precursor sequences, such as those disclosed in SEQ ID NO:1, 2,4, 6 and 81-270 of WO2017109629, which is incorporated herein by reference in its entirety.

The F1 polypeptide and F2 polypeptide into which the mutant RSV F protein or mutants are introduced may be from any wild-type RSV F protein known or discovered in the future in the art, including but not limited to the F protein amino acid sequences of RSV subtype a and subtype B strains (including A2 Ontario and Buenos Aires) or any other subtype. In some embodiments, the RSV F mutant comprises an F1 and/or F2 polypeptide from an RSV a virus in which one or more mutations are introduced, such as the F1 and/or F2 polypeptides from an RSV F0 precursor protein set forth in any one of SEQ ID NOs 1,2, 4,6, and 81-270 from WO2017109629, which sequences are incorporated herein by reference in their entirety. In some other embodiments, the RSV F mutant comprises an F1 and/or F2 polypeptide from an RSV B virus, such as the F1 and/or F2 polypeptide from an RSV F0 precursor protein set forth in any one of SEQ ID NOs 2 and 211-263 of WO2017/109629, which sequences are incorporated herein by reference in their entirety, into which one or more mutations are introduced. In yet other embodiments, the RSV F mutant comprises an F1 and/or F2 polypeptide from an RSV bovine virus in which one or more mutations are introduced, such as the F1 and/or F2 polypeptides from the RSV F0 precursor protein set forth in any of SEQ ID NOS 264-270 of WO2017109629, which sequences are incorporated herein by reference in their entirety.

The term "F0 polypeptide" (F0) refers to a precursor polypeptide of the RSV F protein, which consists of a signal polypeptide sequence, an F1 polypeptide sequence, a pep27 polypeptide sequence, and an F2 polypeptide sequence. In rare exceptions, the F0 polypeptide of the known RSV strain consists of 574 amino acids.

The term "F1 polypeptide" (F1) refers to the polypeptide chain of the mature RSV F protein. Natural F1 comprises approximately residues 137-574 of the RSV F0 precursor and consists (from N-terminus to C-terminus) of the extracellular region (approximately residues 137-524), the transmembrane domain ("TM") (approximately residues 525-550) and the cytoplasmic tail ("CT") (approximately residues 551-574). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides that include modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, e.g., modifications designed to stabilize or enhance the immunogenicity of RSV F protein mutants.

The term "F2 polypeptide" (F2) refers to the polypeptide chain of the mature RSV F protein. Natural F2 comprises approximately residues 26-109 of RSV F0 precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides that include modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, e.g., modifications designed to stabilize or enhance the immunogenicity of a mutant RSV F protein in a pre-fusion configuration. In the native RSV F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer. The term "foldon" or "folding subdomain" refers to an amino acid sequence capable of forming a trimer. One example of such a folding subdomain is a peptide sequence derived from bacteriophage T4 fibrin, which has the sequence of GYIPEAPRDG QAYVRKDGEW VLLSTFL (SEQ ID NO: 45).

In some embodiments, the RNA molecule encodes RSV F protein mutants ：WO2009/079796、WO2010/149745、WO2011/008974、WO2014/160463、WO2014/174018、WO2014/202570、WO2015/013551、WO2015/177312、WO2017/005848、WO2017/174564、WO2017/005844 and WO2018/109220 disclosed below. The RSV F proteins disclosed in these references are incorporated herein by reference in their entirety.

Antibodies to RSV F protein are common after natural infection and after vaccination, and have been shown to neutralize viral activity in vitro. As used herein, the term "respiratory syncytial virus" or "RSV" is not limited to any particular strain or variant.

In some embodiments, the RNA molecule comprises an open reading frame encoding an RSV antigen. In some embodiments, the RSV antigen is an RSV polypeptide. In some embodiments, the RSV polypeptide is an RSV glycoprotein or a fragment or variant thereof. In some embodiments, the RNA molecule encodes an RSV F protein.

In some embodiments, the RSV polypeptide is a full length RSV polypeptide. In some embodiments, the RSV polypeptide is a truncated RSV polypeptide. In some embodiments, the RSV polypeptide is a variant of an RSV polypeptide. In some embodiments, the RSV polypeptide is a fragment of an RSV polypeptide.

In some embodiments, the RSV polypeptide is a full length RSV F protein. In some embodiments, the RSV polypeptide is a truncated RSV F protein. In some embodiments, the RSV polypeptide is a variant of the RSV F protein. In some embodiments, the RSV polypeptide is a fragment of the RSV F protein.

In some embodiments, the RSV F protein comprises at least one mutation. In some embodiments, the RSV F protein comprises at least two mutations. In some embodiments, the RSV F protein comprises at least three mutations. In some embodiments, the RSV F protein comprises at least four mutations. In some embodiments, the RSV F protein comprises 4 mutations. In some embodiments, the RSV F protein comprises at least five mutations.

In some embodiments, the RNA molecule encodes an RSV F protein set forth in table 1 (see example 6). In some embodiments, the RNA molecule encodes an RSV F protein comprising the amino acid sequence of any of SEQ ID NOs 1-6 and 71-74, or a fragment or variant thereof. In some embodiments, the RSV F polypeptide may have at least, up to, just below, or between any two of the amino acid sequences of Table 1 (e.g., any of SEQ ID NOS: 1-6 and 71-74) identity ：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%. In some embodiments, the RSV F protein consists of any of the amino acid sequences of Table 1 (e.g., any of SEQ ID NOS: 1-6 and 71-74).

In some embodiments, the RNA molecule sequence is transcribed from the DNA nucleic acid sequence (DNA polynucleotide) of table 2 (see example 6). In some embodiments, the RNA molecule comprises an ORF transcribed from the nucleic acid sequence of any one of SEQ ID NOS: 7-10 and 59-62, or a fragment or variant thereof. In some embodiments, the RNA molecule comprises ORF：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% of a nucleic acid sequence transcribed from a nucleic acid sequence that may have at least, up to, just below, or between any two of the following identities with any of the nucleic acid sequences of Table 2 (e.g., any of SEQ ID NOS: 7-10 and 59-62). In some embodiments, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any of the nucleic acid sequences of Table 2 (e.g., any of SEQ ID NOS: 7-10 and 59-62).

In some embodiments, the RNA molecule comprises an ORF comprising the RNA nucleic acid sequence (RNA polynucleotide) of table 3 (see example 6). In some embodiments, the RNA molecule comprises an ORF comprising the nucleic acid sequence of any one of SEQ ID NOS 11-16 and 63-70, or a fragment or variant thereof. In some embodiments, the RNA molecule comprises ORF：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% of a nucleic acid sequence that may have at least, up to, just below, or between any two of the following identities with any of the RNA nucleic acid sequences of Table 3 (e.g., any of SEQ ID NOS: 11-16 and 63-70). In some embodiments, the RNA molecule comprises an ORF that comprises a nucleic acid sequence consisting of any one of the RNA nucleic acid sequences of Table 3 (e.g., any one of SEQ ID NOS: 11-16 and 63-70).

In some embodiments, the RNA molecule comprises stabilized RNA. In some embodiments, the RNA molecule comprises at least one uridine N1-methyl pseudouridine substituted nucleic acid sequence. In some embodiments, the RNA molecule comprises a sequence (denoted as "ψ") of all uridine replaced with N1-methyl pseudouridine. In some embodiments, the RNA molecule comprises an ORF that comprises the nucleic acid sequence of any one of SEQ ID NOs 11-16 and 63-70, wherein all uridine has been replaced with N1-methyl pseudouridine (denoted as "ψ").

In some embodiments, the RNA molecule comprises an open reading frame encoding an amino acid sequence of the RSV F protein that is identical to, at least, up to, just below, or between any two of SEQ ID NO's 1-6 and 71-74 (Table 1) or any of the other RSV F protein fusion pre-F proteins described herein, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some embodiments, the RNA molecule comprises an open reading frame encoding an amino acid sequence of the RSV F protein consisting of any of SEQ ID NO: 1-6 and 71-74 (Table 1) or other RSV pre-fusion F protein described herein.

In some embodiments, the RNA molecule comprises an open reading frame 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% transcribed from a DNA nucleic acid sequence that is identical to any one of the nucleic acid sequences of SEQ ID NOs 7-10 and 59-62 (Table 2) or to any two of the other nucleic acids described herein. In some embodiments, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence consisting of any of the nucleic acid sequences of SEQ ID NOs 7-10 and 59-62 (Table 2) or other nucleic acids described herein.

In some embodiments, the RNA molecules comprise an open reading frame comprising an RNA nucleic acid sequence that is identical to any of the nucleic acid sequences of SEQ ID NOs 11-16 and 63-70 (Table 3) or any of the other nucleic acids described herein, at least, up to, just below, or between any two of 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence consisting of any one of the nucleic acid sequences of SEQ ID NOs 11-16 and 63-70 (Table 3) or other nucleic acids described herein. In some embodiments, the RNA molecule comprises an ORF that comprises the nucleic acid sequence of any one of SEQ ID NOs 11-16 and 63-70 (Table 3), wherein all uridine has been replaced with N1-methyl pseudouridine (denoted as "ψ").

[ III. RNA molecules ]

In some embodiments, the RNA molecules described herein are coding RNA molecules. Coding RNAs include functional RNA molecules that can be translated into peptides or polypeptides. In some embodiments, the coding RNA molecule includes at least one Open Reading Frame (ORF) encoding at least one peptide or polypeptide. The open reading frame comprises a codon sequence translatable into a peptide or protein. The coding RNA molecule may comprise one (monocistronic), two (bicistronic) or more (polycistronic) ORFs, which may be codon sequences translatable into a polypeptide or protein of interest.

The coding RNA molecule may be a messenger RNA (mRNA) molecule, a viral RNA molecule or a self-amplifying RNA molecule (saRNA, also known as a replicon). In some embodiments, the RNA molecule is mRNA. Preferably, the RNA molecule of the invention is mRNA. In some embodiments, the RNA molecule is modRNA. In some embodiments, the RNA molecule is saRNA. In some embodiments, the saRNA molecule may be a coding RNA molecule.

The RNA molecule may encode one polypeptide of interest or more, such as one antigen or more than one antigen, for example two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively or additionally, one RNA molecule may also encode more than one polypeptide of interest, such as an antigen, e.g. a bicistronic or tricistronic RNA molecule encoding a different or the same antigen.

The sequence of the RNA molecule may be codon optimized or de-optimized for expression in a desired host, such as a human cell. In some embodiments, the gene of interest (e.g., antigen) described herein is encoded by a coding sequence that is codon optimized and/or has an increased guanosine/cytidine (G/C) content as compared to the wild-type coding sequence. In some embodiments, one or more sequence regions of the coding sequence are codon optimized and/or have an increased G/C content as compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, codon optimization and/or increasing G/C content does not alter the sequence of the encoded amino acid sequence.

It will be appreciated by those skilled in the art that the term "codon optimization" refers to altering codons in the coding region of a nucleic acid molecule to reflect typical codon usage of the host organism without altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present invention, in some embodiments, the coding region is codon optimized for optimal expression in an individual to be treated with an RNA polynucleotide described herein. Codon optimization is based on the finding that translation efficiency is also determined by the frequency of occurrence of tRNA molecules in cells. Thus, the sequence of the RNA can be modified such that frequently occurring codons of the tRNA molecule are inserted at the "rare codon" positions.

In some embodiments, the G/C content of the coding region (e.g., of the gene sequence of interest; open Reading Frame (ORF)) of the RNA is increased as compared to the G/C content of the corresponding coding sequence of a wild-type RNA encoding the gene of interest, wherein in some embodiments the amino acid sequence encoded by the RNA is unmodified as compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of the mRNA. The sequence with increased G (guanosine)/C (cytidine) content is more stable than the sequence with increased A (adenosine)/U (uridine) content. The codon most advantageous for stability (so-called substitution codon usage) can be determined with respect to the fact that several codons encode the same amino acid (so-called degeneracy of the genetic code). Depending on the amino acids encoded by the RNA, there are various possibilities for modification of the RNA sequence compared to its wild-type sequence. In particular, codons containing a and/or U nucleosides can be modified by replacing these codons with other codons encoding the same amino acid but not containing a and/or U or containing a lower content of a and/or U nucleosides. Thus, in some embodiments, the G/C content of the coding region of an RNA described herein is increased by at least, up to, just below, or between any two of 10%, 20%, 30%, 40%, 50%, 55%, or even more, as compared to the G/C content of the coding region of a wild-type RNA. In some embodiments, the coding region of an RSV RNA described herein comprises a G/C content of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or about 80%. In some embodiments, the coding region of RSV RNA described herein comprises a G/C content of about 50% -75%, about 55% -70%, about 50% -60%, about 60% -70%, about 70% -80%, about 50% -55%, about 55% -60%, about 60% -65%, about 65% -70%, about 70% -75%, or about 75% -80%. In some embodiments, the coding region of an RSV RNA described herein comprises a G/C content of about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74% or about 75%. In some embodiments, the coding region of an RSV RNA described herein comprises a G/C content of about 58%, about 66%, or about 62%.

In some embodiments, the RNA molecule comprises about 20 to about 100,000 nucleotides (e.g., 30～50、30～100、30～250、30～500、30～1,000、30～1,500、30～3,000、30～5,000、30～7,000、30～10,000、30～25,000、30～50,000、30～70,000、100～250、100～500、100～1,000、100～1,500、100～3,000、100～5,000、100～7,000、100～10,000、100～25,000、100～50,000、100～70,000、100～100,000、500～1,000、500～1,500、500～2,000、500～3,000、500～5,000、500～7,000、500～10,000、500～25,000、500～50,000、500～70,000、500～100,000、1,000～1,500、1,000～2,000、1,000～3,000、1,000～5,000、1,000～7,000、1,000～10,000、1,000～25,000、1,000～50,000、1,000～70,000、1,000～100,000、1,500～3,000、1,500～5,000、1,500～7,000、1,500～10,000、1,500～25,000、1,500～50,000、1,500～70,000、1,500～100,000、2,000～3,000、2,000～5,000、2,000～7,000、2,000～10,000、2,000～25,000、2,000～50,000、2,000～70,000、 and 2,000 to 100,000 nucleotides).

In some embodiments, the RNA molecule has at least, up to, just below, or between any two of about 20、40、60、80、100、120、140、160、180、200、220、240、260、280、300、320、340、360、380、400、420、440、460、480、500、520、540、560、580、600、620、640、660、680、700、720、740、760、780、800、820、840、860、880、900、920、940、960、980、1000、1200、1400、1600、1800、2000、2200、2400、2600、2800、3000、3200、3400、3600、3800、4000、4200、4400、4600、4800、5000、5200、5400、5600、5800、6000、6200、6400、6600、6800、7000、7200、7400、7600、7800、8000、8200、8400、8600、8800、9000、9200、9400、9600、9800、10000、12000、14000、16000、18000、20000、22000、24000、26000、28000、30000、32000、34000、36000、38000、40000、42000、44000、46000、48000、50000、52000、54000、56000、58000、60000、62000、64000、66000、68000、70000、72000、74000、76000、78000、80000、82000、84000、86000、88000、90000、92000、94000、96000、98000 or 100000 nucleotides.

In some embodiments, the RNA molecule comprises at least 100 nucleotides. For example, in some embodiments, the RNA is between 100 and 15,000 nucleotides in length, between 7,000 and 16,000 nucleotides, between 8,000 and 15,000 nucleotides, between 9,000 and 12,500 nucleotides, between 11,000 and 15,000 nucleotides, between 13,000 and 16,000 nucleotides, and between 7,000 and 25,000 nucleotides. In some embodiments, the RNA molecule has at least, up to, just below, or between any two of about 100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250、1300、1350、1400、1450、1500、1550、1600、1650、1700、1750、1800、1850、1900、1950、2000、2050、2100、2150、2200、2250、2300、2350、2400、2450、2500、2550、2600、2650、2700、2750、2800、2850、2900、2950、3000、3050、3100、3150、3200、3250、3300、3350、3400、3450、3500、3550、3600、3650、3700、3750、3800、3850、3900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450、5500、5550、5600、5650、5700、5750、5800、5850、5900、5950、6000、6050、6100、6150、6200、6250、6300、6350、6400、6450、6500、6550、6600、6650、6700、6750、6800、6850、6900、6950、7000、7050、7100、7150、7200、7250、7300、7350、7400、7450、7500、7550、7600、7650、7700、7750、7800、7850、7900、7950、8000、8050、8100、8150、8200、8250、8300、8350、8400、8450、8500、8550、8600、8650、8700、8750、8800、8850、8900、8950、9000、9050、9100、9150、9200、9250、9300、9350、9400、9450、9500、9550、9600、9650、9700、9750、9800、9850、9900、9950、10000、10050、10100、10150、10200、10250、10300、10350、10400、10450、10500、10550、10600、10650、10700、10750、10800、10850、10900、10950、11000、11050、11100、11150、11200、11250、11300、11350、11400、11450、11500、11550、11600、11650、11700、11750、11800、11850、11900、11950、12000、12050、12100、12150、12200、12250、12300、12350、12400、12450、12500、12550、12600、12650、12700、12750、12800、12850、12900、12950、13000、13050、13100、13150、13200、13250、13300、13350、13400、13450、13500、13550、13600、13650、13700、13750、13800、13850、13900、13950、14000、14050、14100、14150、14200、14250、14300、14350、14400、14450、14500、14550、14600、14650、14700、14750、14800、14850、14900、14950 or 15000 nucleotides.

The RNA molecules of the invention can be prepared by any method known in the art, including chemical synthesis and in vitro methods, such as RNA in vitro transcription. In some embodiments, the RNA lines of the invention are prepared using in vitro transcription.

In some embodiments, the RNA molecules of the invention are purified, for example, by filtration, which may be performed via, for example, ultrafiltration, diafiltration, or, for example, tangential flow ultrafiltration/diafiltration.

In some embodiments, the RNA molecules of the invention are lyophilized to be temperature stable.

In some embodiments of the invention, the RNA is or comprises messenger RNA (mRNA) associated with an RNA transcript encoding a polypeptide. In some embodiments, the RNAs disclosed herein comprise a 5 'cap comprising a 5' cap disclosed herein, a 5 'untranslated region (5' utr) comprising a cap proximal sequence, a sequence encoding a protein (e.g., a polypeptide) (e.g., an F protein prior to RSV fusion), a3 'untranslated region (3' utr), and/or a polyadenylation (poly a) sequence.

In some embodiments, the RNAs disclosed herein comprise, in a 5 'to 3' orientation, a 5 'cap comprising a 5' cap disclosed herein, a 5 'untranslated region (5' utr) comprising a cap proximal sequence, a sequence encoding a protein (e.g., a polypeptide) (e.g., a pre-RSV fusion F protein), a 3 'untranslated region (3' utr), and a poly-a sequence.

In some embodiments, the RNAs disclosed herein further comprise a signal peptide. Non-limiting examples of signal peptides, as well as amino acid and nucleic acid sequences encoding such peptides, can be found, for example, in WO2017/109629 (the disclosure of which is incorporated herein by reference in its entirety).

In some embodiments, the RNAs disclosed herein encode antigenic fusion proteins. Thus, the encoded antigen or antigens may include two or more proteins (e.g., proteins and/or protein fragments) that are joined together. Or a protein fused to a protein antigen does not promote a strong immune response against itself, but rather against the antigen. In some embodiments, the antigenic fusion proteins retain the functional properties of each of the original proteins. In some embodiments, the RNAs disclosed herein encode fusion proteins comprising an antigen linked to a backbone moiety. In some embodiments, the RNA further encodes a linker located between at least one or each domain of the fusion protein. Non-limiting examples of such backbone moieties and linkers can be found, for example, in WO 2022/067010 (the disclosure of which is incorporated herein by reference in its entirety).

[ A ] modified nucleobases ]

In some embodiments of the invention, the RNA molecule is not chemically modified and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the invention comprise standard nucleoside residues, such as those present in the transcribed RNA (e.g., A, G, C and/or U). In some embodiments, the nucleotides and nucleosides of the invention comprise standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, and/or dT).

In other embodiments of the invention, the RNA molecules can comprise modified nucleobases, which can be incorporated into modified nucleosides and nucleotides. In some embodiments, the RNA molecule can include one or more modified nucleotides. In some embodiments, the RNA molecule can include one or more modified nucleotides. Naturally occurring nucleotide modifications are known in the art. In some embodiments, the RNA molecule can include modified nucleotides. Non-limiting examples of modified nucleotides that may be included in the RNA molecule include pseudouridine, N1-methyl pseudouridine, 5-methyl uridine, 3-methyl-uridine, 5-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid methyl, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, uridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-aminomethyl-2-thiouridine, 5-methylaminomethyl-uridine, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethyl aminomethyl-2-thiouridine, 5-propargyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine, 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouridine, 5-taurine methyl-2-amino-pseudouridine, 1-taurine methyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-1-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine, 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine, 5- (isopentenylaminomethyl) uridine, 5- (isopentenylaminomethyl) -2-thiouridine, a-thio-uridine, 2' -O-methyl-uridine, 5,2' -O-dimethyl-uridine, 2' -O-methyl-pseudouridine, 2-thio-2 ' -O-methyl-uridine, 5-methoxycarbonylmethyl-2 ' -O-methyl-uridine, 5-carbamoylmethyl-2 ' -O-methyl-uridine, 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine, 3,2' -O-dimethyl-uridine, 5- (isopentenylaminomethyl) -2' -O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2' -F-arabinoside, 2' -F-uridine, 2' -OH-arabinoside, 5- (2-methoxycarbonylvinyl) uridine, 5- [3- (1-E-propenyl amino) uridine, any other modified uridine known in the art, or a combination thereof. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing modified nucleotides may be excluded from the RNA molecules disclosed herein.

Modifications that may be present in the RNA molecule further include, but are not limited to, for example, ms2io6A (2-methylsulfanyl- (N6- (cis-hydroxyisopentenyl) adenosine), ms2m6A (2-methylsulfanyl-N6-methyladenosine), ms2t6A 2-methylsulfanyl-N6-threonyl carbamoyl adenosine, g6A (N6-glycylcarbamoyladenosine), i6A (N6-isopentenyl) adenosine, m6A (N6-methyladenosine), t6A (N6-threonyl carbamoyl adenosine), m ' Am (1, 2' -O-dimethyladenosine), m1A (1-methyladenosine), 2' -O-methyladenosine, ar (p) (2 ' -O-ribosyl adenosine (phosphate)), 2-methylsulfanyl-N6-isopentenyl adenosine, 2hn6A (2-methylsulfanyl-N6-hydroxyisovaleryl adenosine), m6A (N6-methyladenosine), t6A (N6-threonyl carbamoyl adenosine), m ' Am (1, 2' -O-dimethyladenosine), m1A (1, 2' -O-ribosyl adenosine, ar (2 ' -O-ribosyl adenosine), 2' -O-ribosyl adenosine (phosphate), N6-Dimethyladenosine), ac6A (N6-acetyl adenosine), hn6A (N6-hydroxy-N-valylcarbamoyladenosine), m6t6A (N6-methyl-N6-threonyl-adenine), m2A (2-methyladenosine), ms2i6A (2-methylsulfanyl-N6-isopentenyl-adenine), 7-deaza-adenine, N1-methyl-adenosine, N6- (dimethyl) adenine, N6-cis-hydroxy-isopentenyl-adenosine, a-thio-adenosine, 2- (amino) adenine, 2- (aminopropyl) adenine, 2- (methylsulfanyl) -N6- (isopentenyl) adenine, 2- (alkyl) adenine, 2- (amino) adenine, 2- (aminopropyl) adenine, 2- (halo) adenine, 2- (propyl) adenine, 2' -amino-2 ' -deoxy-adenine, 2' -deoxy-2 ' -hydroxy-isopentenyl-adenine, a-thio-adenine, 2' -amino-deoxyadenosine, 2' -amino-6- (methyl) adenine, 2' -amino-deoxyadenosine, 2' -amino-6- (6-alkyl) adenine, 2' -amino-deoxyadenosine, 2- (6-methyl) adenine, 2' -amino-N-6 ' -acyl-N6- (halo) adenine ) Adenine, 8- (alkenyl) adenine, 8- (alkynyl) adenine, 8- (amino) adenine, 8- (sulfanyl) adenine, 8- (alkenyl) adenine, 8- (alkyl) adenine, 8- (alkynyl) adenine, 8- (amino) adenine, 8- (halo) adenine, 8- (hydroxy) adenine, 8- (sulfanyl) adenine, 8- (thiol) adenine, 8-azido-adenine, 8-oxo-adenine, azaadenine, deazaadenine, N6- (methyl) adenine, N6- (isopentyl) adenine, 7-deaza-8-aza-adenosine, 7-methyladenine, 1-deazaadenosine TP, 2 'fluoro-N6-Bz-deoxyadenosine TP, 2' -OMe-2-amino-ATP, 2 'O-methyl-N6-Bz-deoxyadenosine TP, 2' -a-ethynyl adenine, 2-amino-ATP, 2-fluoro-adenosine 2 '-fluoro-2' -aza-adenosine, 2 '-chloro-2' -fluoro-adenosine, 2' -difluoroadenosine TP;2' -deoxy-2 ' -a-mercaptoadenosine TP;2' -deoxy-2 ' -a-thiomethoxy adenosine TP, 2' -deoxy-2 ' -b-amino adenosine TP, 2' -deoxy-2 ' -b-azido adenosine TP, 2' -deoxy-2 ' -b-bromo adenosine TP, 2' -deoxy-2 ' -b-chloro adenosine TP, 2' -deoxy-2 ' -b-fluoro adenosine TP, 2' -deoxy-2 ' -b-iodo adenosine TP, 2' -deoxy-2 ' -b-mercapto adenosine TP, 2' -deoxy-2 ' -b-thiomethoxy adenosine TP, 2-fluoro adenosine TP, 2-iodo adenosine TP, 2-mercapto adenosine TP, 2-methoxy adenine, 2-methylthio-adenine, 2-trifluoromethyl adenosine TP, 3-deaza-3-bromo adenosine TP, 3-deaza-3-chloro adenosine TP, 3-deaza-3-iodo adenosine TP, 4' -azido adenosine carbocyclic adenosine TP, 4' -deoxy-2 ' -b-mercapto adenosine TP, 2' -mercapto adenosine TP, 2-methoxy adenine TP, 2-methylthio adenosine TP, 3-deaza-3-bromo adenosine TP, 3-deaza-3-chloro adenosine TP, 3-deaza-3-chloro adenosine TP, 4' -azido adenosine TP, 4' -thio adenosine TP, 5' -bromo adenosine TP, 8-deaza adenosine TP, 8-2 ' -bromo adenosine TP Azapurines, 7-deaza-7-substituted purines, 7-deaza-8-substituted purines, 7-deaza-2, 6-diaminopurines, 7-deaza-8-aza-2-aminopurine, 2, 4-diaminopurines, 2, 6-diaminopurines, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 8-azapurines, s2C (2-thiocytosine), m3C (3-methylcytidine), f5C (5-formylcytidine), hm5C (5-hydroxymethylcytosine), m5C (5-methylcytidine), ac4C (N4-acetylcytidine), cm (2 ' -O-methylcytidine), m5Cm (5, 2' -O-dimethylcytidine), f5Cm (5-formyl-2 ' -O-methylcytidine), k2C (Lysidine)), m4C (N4-acetylcytidine), n4-dimethyl-2 ' -OMe-cytidine TP, 4-methylcytidine, 5-aza-cytidine, pseudo-iso-cytidine, pyrrolo-cytidine, a-thio-cytidine, 2- (thio) cytosine, 2' -amino-2 ' -deoxy-CTP, 2' -azido-2 ' -deoxy-CTP, 2' -deoxy-2 ' -a-aminocytidine TP; 2' -deoxy-2 ' -a-azidocytidine TP, 3- (deaza) 5- (aza) cytosine, 3- (methyl) cytosine, 3- (alkyl) cytosine, 3- (deaza) 5- (aza) cytosine, 3- (methyl) cytidine, 4,2' -O-dimethylcytidine, 5- (halo) cytosine, 5- (methyl) cytosine, 5- (propynyl) cytosine, 5- (trifluoromethyl) cytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, 5-hydroxycytosine, 5-methylcytosine, 5- (alkyl) cytosine, 5- (alkenyl) cytosine, 5- (alkynyl) cytosine, 5- (halo) cytosine, 5- (propynyl) cytosine, 5- (trifluoromethyl) cytosine, 5-bromo-cytidine, 5-iodo-cytidine, 5-propynyl cytosine, 6- (azo) cytosine, 6-aza-cytidine, N4- (acetyl) cytosine, 1-methyl-cytidine, 1-methoxy-2-isopropyl-cytidine, 5-methoxy-2-methoxy-cytidine, 5-isopropyl-2-methoxy-cytidine 1-methyl-pseudoisocytosine, 4-methoxy-pseudoisocytosine, 4-thio-1-methyl-1-deaza-pseudoisocytosine, 4-thio-1-methyl-pseudoisocytosine, 4-thio-pseudoisocytosine, 5-aza-zebularin (zebularine), 5-methyl-zebularin, pyrrolo-pseudoisocytosine, zebularin (E) -5- (2-bromo-vinyl) cytidine TP, 2 '-anhydro-cytidine TP hydrochloride, 2' -fluoro-N4-Bz-cytidine TP, 2 '-fluoro-N4-acetyl-cytidine TP, 2' -O-methyl-N4-Bz-cytidine TP, 2 '-a-ethynyl cytidine TP, 2' -a-trifluoromethyl cytidine TP, 2 '-b-ethynyl cytidine TP, 2' -b-trifluoromethyl cytidine TP, 2 '-deoxy-2', 2' -difluorocytidine TP;2' -deoxy-2 ' -a-mercaptocytidine TP, 2' -deoxy-2 ' -a-thiomethoxycytidine TP, 2' -deoxy-2 ' -b-aminocytidine TP, 2' -deoxy-2 ' -b-azidocytidine TP, 2' -deoxy-2 ' -b-bromocytidine TP, 2' -deoxy-2 ' -b-chlorophenyl) -2' -b-fluorocytidine TP, 2' -deoxy-2 ' -b-iodocytidine TP, 2' -deoxy-2 ' -b-mercaptocytidine TP, 2' -deoxy-2 ' -b-thiomethoxycytidine TP, 2' -O-methyl-5- (1-propynyl) cytidine TP, 3' -ethynyl cytidine TP, 4' -azidocytidine TP, 4' -acetylenyl cytidine TP, 5- (1-propynyl) arabino-cytidine TP, 5- (2-chloro-phenyl) -2-thiocytidine TP, 5- (4-amino-phenyl) -2-thiocytidine TP, 5-aminopropyl-2 ' -b-mercaptocytidine TP, 2' -deoxy-2 ' -b-thiocytidine TP, 2' -O-methyl-5- (1-propynyl) cytidine TP, 4' -azido-cytidine TP, 5' -ethynyl cytidine TP, 5' -azido cytidine TP, 5-fluoro-cytidine TP Glycoside TP, N4-benzoyl-cytidine TP, pseudoisocytidine, mimG (methylguanosine), m7G (7-methylguanosine), m2Gm (N2, 2' -O-dimethylguanosine), m2G (N2-methylguanosine), imG (Russian glycoside (Wyosine)), m1Gm (1, 2' -O-dimethylguanosine), m1G (1-methylguanosine), 2' -O-methylguanosine, 2' -O-ribosyl guanosine (phosphate), gm (2 ' -O-methylguanosine), gr (p) (2 ' -O-ribosyl guanosine (phosphate)), preQi (7-aminomethyl-7-deammoniaguanosine), preQo (7-cyano-deammoniaguanosine), G (ancient P) and methyl Russian guanosine (Archaeosine), m2'7G (N2, 7-dimethylguanosine), m22Gm (N2, N2,2' -O-methylguanosine), m2' -O-ribosyl guanosine (phosphate), gm (2 ' -O-methylguanosine), gr (2 ' -O-riboguanosine), m (7-riboguanosine) (phosphate)), G (7-cyano-7-deammoniaguanosine), G (N2, N2G 2, N2,7,2' -O-trimethylguanosine, 6-thio-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, N1-methyl-guanosine, a-thio-guanosine, 2- (propyl) guanosine, 2- (alkyl) guanosine, 2' -amino-2 ' -deoxy-GTP, 2' -azido-2 ' -deoxy-GTP, 2' -deoxy-2 ' -a-amino-guanosine TP, 2' -deoxy-2 ' -a-azido-guanosine TP, N2-dimethyl-guanosine, 6- (methyl) guanosine, 6- (alkyl) guanosine, 6- (methyl) guanosine, 6-methyl-guanosine, 6-thioguanosine, 7- (alkyl) guanosine, 7-deaza-7- (C2-C6) alkynylguanine, 7- (methyl) guanosine, 7- (alkyl) guanosine, 7- (nitro) guanosine, 7- (methyl) guanosine, 7- (deaza) guanosine, 7- (methyl) guanosine, 8-aza-guanosine, 8-hydroxy-8- (alkyl) guanosine, 6-thioguanosine, 7- (alkyl) guanosine, 8- (hydroxy) guanosine, 8- (alkyl) guanosine, 8- (methyl) guanosine ) Guanine, 8- (alkynyl) guanine, 8- (amino) guanine, 8- (halo) guanine, 8- (hydroxy) guanine, 8- (sulfanyl) guanine, 8- (thiol) guanine, azaguanine, deazaguanine, N (methyl) guanine, N- (methyl) guanine, 1-methyl-6-thio-guanosine, 6-methoxy-guanosine, 6-thio-7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-methyl-guanosine, 7-deaza-8-aza-guanosine, 7-methyl-8-oxo-guanosine, N2-dimethyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, 1-me-GTP, 2' fluoro-N2-isobutyl-guanosine TP, 2' -O-methyl-N2-isobutyl-guanosine TP, 2' -a-ethynyl-guanosine TP; 2' -a-trifluoromethyl guanosine TP;2' -b-ethynyl guanosine TP;2' -b-trifluoromethyl guanosine TP, 2' -deoxy-2 ',2' -difluoro guanosine TP, 2' -deoxy-2 ' -a-mercapto guanosine TP, 2' -deoxy-2 ' -a-thiomethoxy guanosine TP, 2' -deoxy-2 ' -b-amino guanosine TP, 2' -deoxy-2 ' -b-azido guanosine TP, 2' -deoxy-2 ' -b-bromo guanosine TP, 2' -deoxy-2 ' -b-chloro guanosine TP, 2' -deoxy-2 ' -b-fluoro guanosine TP, 2' -deoxy-2 ' -b-iodo guanosine TP, 2' -deoxy-2 ' -b-mercapto guanosine TP, 2' -deoxy-2 ' -b-thiomethoxy guanosine TP, 4' -azido guanosine TP, 4' -carbo-cyclic guanosine TP, 5' -high-guanosine TP, 8-bromo-guanosine TP, 9-deazaguanosine TP, N2-isobutyl-guanosine TP, 24 (1-methyl inosine) I (1-inosine), 1,2' -dimethyl-2 ' -b-fluoro guanosine TP (2 ' -methyl-82-methyl-inosine) Q (43 oL-O-52), 2' -thio guanosine TP (N-methyl-E-52-O-methyl-inosine) Q (52-E) glycoside-Q nucleoside), manQ (mannosyl Q nucleoside), Q (Q nucleoside), allylamino-thymidine, azathymidine, deazathymidine, deoxythymidine, um (2 '-O-methyluridine), s2U (2-thiouridine), m3U (3-methyluridine), cm5U (5-carboxymethyluridine), ho5U (5-hydroxyuridine), m5U (5-methyluridine), tm5s2U (5-taurinomethyl-2-thiouridine), 5-taurinomethyl uridine, D (dihydrouridine), pseudouridine, acp3U (3- (3-amino-3-carboxypropyl) uridine), 1-methyl-3- (3-amino-5-carboxypropyl) pseudouridine, 1-methyl pseudouridine, 1-ethyl-pseudouridine, 2' -O-methyluridine, s2U (2-thiouridine), tm5 '-O-2' -methyluridine, m (3-carboxyuridine), 3- (3-amino-3-carboxypropyl) uridine, acp3U (3-amino-3-carboxypropyl) uridine, 1-methyl pseudouridine, 2 '-O-methyluridine, s2U (2-thiouridine), m 2' -O-methyluridine, m (5-thiouridine, m (3-methyluridine), 3-amino-3- (3-amino-carboxypropyl) uridine, 3-methyl-3-carboxypropyl) uridine; m 5-Um (5, 2 '-O-dimethyluridine), 5, 6-dihydro-uridine, nm5s2U (5-aminomethyl-2-thiouridine), ncm5U (5-carbamoylmethyl-2' -O-methyluridine), ncm U (5-carbamoylmethyluridine), 5-carboxyhydroxymethyl uridine methyl ester, cnmm U (5-carboxymethylaminomethyl-2 '-O-methyluridine), cmnm s2U (5-carboxymethylaminomethyl-2-thiouridine), 5-carboxymethylaminomethyluridine, cmnm U (5-carboxymethylaminomethyluridine), 5-carbamoylmethyluridine TP, mcm5U (5-methoxycarbonylmethyl-2' -O-methyluridine), mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine), mcm5U (5-methoxycarbonylmethyluridine), mo5U (5-methoxyuridine), 5-carboxymethyl uridine, 5-U (5-carboxymethyl-2-thiouridine), 5-carboxymethyl-N-methyluridine, 5-carboxymethyl-2-N-methyluridine, 5-carboxymethyl-N-methyluridine, 5-carbamoylmethyluridine, 5-carboxymethyl-2-O-thiouridine, 5-carboxymethyl-N-methyluridine, 5-carbamoylmethyluridine, 5-N-methyluridine Methyl-pseudo-uracil, N1-ethyl-pseudo-uracil, cmo5U (uridine 5-oxyacetic acid), mcmo U (uridine 5-oxyacetic acid methyl ester), 3- (3-amino-3-carboxypropyl) -uridine TP, 5- (iso-pentenylaminomethyl) -2-thiouridine TP, 5- (iso-pentenylaminomethyl) -2' -O-methyluridine TP, 5- (iso-pentenylaminomethyl) uridine TP, 5-propynyluracil, a-thio-uridine, 1- (aminoalkylamino-carbonylamino-vinyl) -2 (thio) -pseudouracil, 1- (aminoalkylamino-carbonylamino-carbonyl vinyl) -2,4- (dithio) pseudouracil, 1- (aminoalkylamino-carbonyl vinyl) -4- (thio) pseudouracil, 1- (aminocarbonyl vinyl) -2 (thio) -pseudouracil, 1- (aminocarbonyl vinyl) -2,4- (dithio) pseudouracil, 1- (aminocarbonyl) -4- (thio) pseudouracil, 1- (amino-carbonyl) -2- (thio) pseudouracil, 1- (thio) -2- (thio) substituted pseudouracil, 1- (amino-carbonyl) -2- (thio) uracil Pseudouracil, 1-substituted 4- (thio) pseudouracil, 1-substituted pseudouracil, 1- (aminoalkylamino-carbonyl vinyl) -2- (thio) -pseudouracil, 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine TP, 1-methyl-3- (3-amino-3-carboxypropyl) pseudoUTP, 1-methyl-pseudoUTP, 1-ethyl-pseudoUTP, 2- (thio) pseudouracil, 2' deoxyuridine, 2' fluorouridine, 2- (thio) uracil, 2,4- (dithio) pseudouracil, 2' methyl, 2 'amino, 2' azido, 2' fluoro-guanosine; 2' -amino-2 ' -deoxy-UTP; 2' -azido-2 ' -deoxy-UTP; 2' -azido-deoxyuridine TP, 2' -O-methylpseudouridine, 2' -deoxyuridine, 2' -fluorouridine, 2' -deoxy-2 ' -a-aminouridine TP, 2' -deoxy-2 ' -a-azido-uridine TP, 2-methylpseudouridine, 3- (3-amino-3-carboxypropyl) uracil, 4- (thio) pseudouracil, 4- (thio) uracil, 4-thiouracil, 5-aminouracil, 5- (1, 3-diazole-1-alkyl) uracil, 5- (2-aminopropyl) uracil, 5- (aminoalkyl) uracil, 5- (dimethylaminoalkyl) uracil, 5- (guanidinoalkyl) uracil, 5- (methoxycarbonylmethyl) -2- (thio) uracil, 5- (methoxycarbonyl-methyl) uracil, 5- (methyl) 2- (thio) uracil, 5- (methyl) 2,4- (dithio) uracil, 5- (methyl) -4- (thio) uracil, 5- (methylaminomethyl) -2- (methyl) uracil, 5- (methylamino) uracil Methyl) -2,4- (dithio) uracil, 5- (methylaminomethyl) -4- (thio) uracil, 5- (propynyl) uracil, 5- (trifluoromethyl) uracil, 5- (2-aminopropyl) uracil, 5- (alkyl) -2- (thio) pseudouracil, 5- (alkyl) -2,4- (dithio) pseudouracil, 5- (alkyl) -4- (thio) pseudouracil, 5- (alkyl) uracil, 5- (alkenyl) uracil, 5- (alkynyl) uracil, 5- (allylamino) uracil, 5- (cyanoalkyl) uracil, 5- (dialkylaminoalkyl) uracil, 5- (dimethylaminoalkyl) uracil, 5- (guanidinoalkyl) uracil, 5- (halo) uracil, 5- (1, 3-diazol-1-alkyl) uracil, 5- (methoxycarbonylmethyl) -2- (thio) uracil, 5- (methoxycarbonyl-methyl) uracil, 5- (thio) uracil, 5- (methyl) uracil, 5- (4-dimethyl) uracil, 5- (4-thio) uracil Sulfur) uracil; 5- (methyl) -2- (thio) pseudouracil; 5- (methyl) -2,4- (dithio) pseudouracil, 5- (methyl) -4- (thio) pseudouracil, 5- (methyl) pseudouracil, 5- (methylaminomethyl) -2- (thio) uracil, 5- (methylaminomethyl) -2,4 (dithio) uracil, 5- (methylaminomethyl) -4- (thio) uracil, 5- (propynyl) uracil, 5- (trifluoromethyl) uracil, 5-aminoallyl-uridine, 5-bromo-uridine, 5-iodo-uridine, 5-uracil, 6- (azo) uracil, 6-aza-uridine, allylamino-uracil, azauracil, deazauracil, 5-methyluracil, 5- (hydroxymethyl) uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, N3- (methyl) uracil, pseudo-UTP-1-2-acetic acid, pseudo-uracil, 4-thio-pseudo-UTP, 1-carboxymethyl-uridine, 1-fluoro-uridine, 1-methyl-1-uridine 1-nitro-1-methyl-uridine 1-nitro-uridine -taurine methyl-4-thio-uridine, 1-taurine methyl-pseudouridine, 2-methoxy-4-thio-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, (±) 1- (2-hydroxypropyl) pseudouridine TP, (2R) -1- (2-hydroxypropyl) pseudouridine TP, (E) -5- (2-bromo-vinyl) arabino-uridine TP, (E) -5- (2-bromo-vinyl) uridine, (Z) -5- (2-bromo-2-vinyl) uridine, (Z) -5- (2, 3-bromo-3, 3-fluoro-ethyl) pseudouridine, (2-3-bromo-3-fluoro-3-vinyl) penta-uridine ) Pseudouridine TP, 1- (2, 2-diethoxyethyl) pseudouridine TP, 1- (2, 4, 6-trimethylbenzyl) pseudouridine TP, 1- (2, 4, 6-trimethyl-benzyl) pseudoUTP, 1- (2, 4, 6-trimethyl-phenyl) pseudoUTP, 1- (2-amino-2-carboxyethyl) pseudoUTP, 1- (2-amino-ethyl) pseudoUTP, 1- (2-hydroxyethyl) pseudouridine TP, 1- (2-methoxyethyl) pseudouridine TP, 1- (3, 4-bis-trifluoromethoxybenzyl) pseudouridine TP, 1- (3, 4-dimethoxybenzyl) pseudouridine TP, 1- (3-amino-3-carboxypropyl) pseudoUTP, 1- (3-amino-propyl) pseudoUTP, 1- (3-cyclopropyl-prop-2-ynyl) pseudouridine TP, 1- (4-amino-4-carboxybutyl) pseudoUTP, 1- (4-amino-ethyl) pseudoUTP, 1- (4-benzyl) pseudouridine TP, 1- (4-bromobenzyl) pseudouridine TP, 1- (3, 4-dimethoxybenzyl) pseudouridine TP, 1- (4-amino-3-carboxypropyl) pseudoUTP, 1- (4-cyclopropyl-prop-2-ynyl) pseudouridine TP, 1- (4-amino-4-carboxybutyl) pseudouridine TP, 1- (4-amino-4-benzyl) uridine TP, 1- (4-trifluoromethyl) and 3-trifluoromethyl) uridine TP ) Pseudouridine TP, 1- (4-fluorobenzyl) pseudouridine TP, 1- (4-iodobenzyl) pseudouridine TP, 1- (4-methylsulfonylbenzyl) pseudouridine TP, 1- (4-methoxybenzyl) pseudouridine TP, 1- (4-methoxy-benzyl) pseudouridine TP, 1- (4-methoxy-phenyl) pseudoUTP, 1- (4-methylbenzyl) pseudouridine TP, 1- (4-methyl-benzyl) pseudoUTP, 1- (4-nitrobenzyl) pseudouridine TP, 1- (4-nitro-benzyl) pseudoUTP, 1 (4-nitro-phenyl) pseudoUTP, 1- (4-thiomethoxybenzyl) pseudouridine TP, 1- (4-trifluoromethoxybenzyl) pseudouridine TP; 1- (4-Trifluoromethylbenzyl) pseudouridine TP, 1- (5-amino-pentyl) pseudoUTP, 1- (6-amino-hexyl) pseudoUTP, 1, 6-dimethyl-pseudoUTP, 1- [3- (2- {2- [2- (2-aminoethoxy) -ethoxy ] -ethoxy) -propionyl ] pseudouridine TP, 1- {3- [2- (2-aminoethoxy) -ethoxy ] -propionyl } pseudouridine TP, 1-acetyl pseudouridine TP, 1-alkyl-6- (1-propynyl) -pseudoUTP, 1-alkyl-6- (2-propynyl) -pseudoUTP, 1-alkyl-6-allyl-pseudoUTP, 1-alkyl-6-ethynyl-pseudoUTP, 1-alkyl-6-homoallyl-pseudoUTP, 1-alkyl-6-vinyl-pseudoUTP, 1-allyluridine TP, 1-aminomethyl-pseudoUTP, 1-benzoyl pseudouridine, 1-methyl-6- (1-propynyl) -pseudouridine TP, 1-alkyl-6-allyl-pseudoUTP, 1-alkyl-6-ethynyl-pseudoUTP, 1-alkyl-6-homoallyl-pseudoUTP, 1-alkyl-6-vinyl-pseudoUTP, 1-aminomethyl-pseudouridine TP, 1-benzyl-P, 1-benzyl-pseudouridine, 1-methyl-P, 1-methyl-6-methyl-p, 1-acetyl pseudouridine, 1-methyl-P, 1-methyl-n-methyl-6-n cyclobutylmethyl-pseudo-UTP, 1-cyclobutyl-pseudo-UTP, 1-cycloheptylmethyl-pseudo-UTP, 1-cycloheptyl-pseudo-UTP, 1-cyclohexylmethyl-pseudo-UTP, 1-cyclohexyl-pseudo-UTP, 1-cyclooctylmethyl-pseudo-UTP, 1-cyclooctyl-pseudo-UTP, 1-cyclopentylmethyl-pseudo-UTP, 1-cyclopentyl-pseudo-UTP, 1-cyclopropylmethyl-pseudo-UTP, 1-cyclopropyl-pseudo-UTP, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudo-uridine TP, 1-hydroxymethyl pseudo-uridine TP, 1-isopropyl-pseudo-UTP, 1-me-2-thio-pseudo-UTP, 1-me-4-thio-pseudo-UTP, 1-me-alpha-thio-pseudo-UTP, 1-methylsulfonylmethyl pseudo-UTP, 1-methoxymethyl pseudo-uridine TP, 1-methoxymethyl pseudo-uridine, 1-ethyl-pseudo-UTP, 1-hexyl-pseudo-UTP, 1-homoallylpseudouridine TP, 1-hydroxymethyl-6- (1-isopropyl-pseudo-UTP), 1-me-2-thio-pseudo-UTP, 1-methyl-alpha-pseudo-UTP, 1-methylsulfonylmethyl-uridine TP, 1-me-4-methyl-4-thio-6- (6-methyl-6-p)) -6-methyl-pseudo-UTP amino-pseudo-UTP, 1-methyl-6-azido-pseudo-UTP, 1-methyl-6-bromo-pseudo-UTP, 1-methyl-6-butyl-pseudo-UTP, 1-methyl-6-chloro-pseudo-UTP, 1-methyl-6-cyano-pseudo-UTP, 1-methyl-6-dimethylamino-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-carboxylate-pseudo-UTP, 1-methyl-6-ethyl-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-formyl-pseudo-UTP, 1-methyl-6-hydroxy-pseudo-UTP, 1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-isopropyl-pseudo-UTP, 1-methyl-6-methoxy-pseudo-UTP, 1-methyl-6-ethoxy-pseudo-UTP, 1-methyl-6-fluoro-pseudo-UTP, 1-methyl-6-hydroxy-pseudo-UTP, 1-methyl-6-iodo-pseudo-UTP, 1-methyl-6-isopropyl-pseudo-UTP, tert-butyl-UTP, 1-methyl-6-fluoro-pseudo-UTP UTP, 1-methyl-6-trifluoromethyl-pseudo-UTP, 1- (N-morpholinyl) methyl pseudouridine TP, 1-pentyl-pseudo-UTP, 1-phenyl-pseudo-UTP, 1-trimethylacetyl pseudouridine TP, 1-propargyl pseudouridine TP, 1-propyl-pseudo-UTP, 1-propynyl-pseudouridine, 1-p-tolyl-pseudo-UTP, 1-tert-butyl-pseudo-UTP, 1-thiomethoxymethyl pseudouridine TP, 1-thio (N-morpholinyl) methyl pseudouridine TP, 1-trifluoroacetyl pseudouridine TP, 1-trifluoromethyl-pseudo-UTP, 1-vinyl pseudouridine TP, 2' -anhydro-uridine TP, 2' -bromo-deoxyuridine TP, 2' -F-5-methyl-2 ' -deoxy-UTP, 2' -OMe-5-me-UTP, 2' -OMe-pseudo-UTP, 2' -a-ethynyl uridine TP, 2' -a-uridine TP, 2' -trifluoromethyl-uridine TP, 2' -fluoro-b-trifluoromethyl-uridine TP, 2' -fluoro-uridine TP, 2' -difluorouridine TP;2' -deoxy-2 ' -a-mercaptouridine TP;2' -deoxy-2 ' -a-thiomethoxyuridine TP, 2' -deoxy-2 ' -b-aminouridine TP, 2' -deoxy-2 ' -b-azido uridine TP, 2' -deoxy-2 ' -b-bromouridine TP, 2' -deoxy-2 ' -b-chlorouridine TP, 2' -deoxy-2 ' -b-fluorouridine TP, 2' -deoxy-2 ' -b-iodouridine TP, 2' -deoxy-2 ' -b-mercaptouridine TP, 2' -deoxy-2 ' -b-thiomethoxyuridine TP, 2-methoxy-4-thio-uridine TP, 2-methoxyuridine, 2' -O-methyl-5- (1-propynyl) uridine TP, 3-alkyl-pseudo-UTP, 4' -azido uridine TP, 4' -carbocycle uridine TP, 5- (1-propynyl) arabino-uridine TP, 5- (2-furanyl) uridine TP, 5-cyanouridine TP, 5-dimethylaminouridine TP, 5' -hyperbaric-2 ' -b-mercaptouridine TP, 2' -O-thiouridine TP, 2' -O-methyl-5- (1-propynyl) uridine TP, 3-alkyl-pseudo-UTP, 4' -azido uridine TP, 5' -fluorouridine TP, 5' -hyperbaric-fluorouridine TP, 5' -fluorouridine TP, 6-fluoro-2 ' -thiouridine TP TP, 5-vinyl-arabinoside TP, 6- (2, 2-trifluoroethyl) -pseudo-UTP, 6- (4- (N-morpholinyl)) -pseudo-UTP, 6- (4-thio (N-morpholinyl)) -pseudo-UTP, 6- (substituted phenyl) -pseudo-UTP, 6-amino-pseudo-UTP, 6-azido-pseudo-UTP, 6-bromo-pseudo-UTP, 6-butyl-pseudo-UTP, 6-chloro-pseudo-UTP, 6-cyano-pseudo-UTP, 6-dimethylamino-pseudo-UTP, 6-ethoxy-pseudo-UTP, 6-carboxylic acid ethyl-pseudo-UTP, 6-fluoro-pseudo-UTP; 6-formyl-pseudo-UTP, 6-hydroxyamino-pseudo-UTP, 6-hydroxy-pseudo-UTP, 6-iodo-pseudo-UTP, 6-isopropyl-pseudo-UTP, 6-methoxy-pseudo-UTP, 6-methylamino-pseudo-UTP, 6-methyl-pseudo-UTP, 6-phenyl-pseudo-UTP, 6-propyl-pseudo-UTP, 6-tert-butyl-pseudo-UTP, 6-trifluoromethoxy-pseudo-UTP, 6-trifluoromethyl-pseudo-UTP, alpha-thio-pseudo-UTP, pseudouridine 1- (4-methylbenzenesulfonic acid) TP, pseudouridine 1- [3- (2-ethoxy) ] propionic acid, pseudouridine 1- [3- {2- (2- [2- (2-ethoxy) -ethoxy ] -ethoxy } propionic acid, pseudouridine 1- [3- {2- (2-ethoxy) -ethoxy ] -ethoxy } propionic acid, alpha-thio-pseudo-uridine 1- [3- (4-methylbenzenesulfonic acid) TP, pseudouridine 1- [3- (2-ethoxy) ] propionic acid, pseudouridine 1- [3- {2- (2-ethoxy) -ethoxy } - [ 2-ethoxy ] -propionic acid - (2-ethoxy) -ethoxy ] propionic acid, pseudouridine TP 1-methylphosphonic acid, diethyl pseudouridine TP 1-methylphosphonate, pseudoUTP-N1-3-propionic acid, pseudoUTP-N1-4-butyric acid, pseudoUTP-N1-5-valeric acid, pseudoUTP-N1-6-hexanoic acid, pseudoUTP-N1-7-heptanoic acid, pseudoUTP-N1-methyl-p-benzoic acid, pseudoUTP-N1-p-benzoic acid, yW (Wybutosine)), OHyW (hydroxy-hubutadine), imG (isobornside), o2yW (peroxy-hubutadine), OHyW (undersodified hydroxy-hubutadine), imG-14 (4-desmethylhumaterial), 2,6- (diamino) purine, 1- (aza) -2- (thio) -3- (aza) -phenazine-1, 3- (aza) -2, 3- (2-oxo) -2- (2, 5-dihydro) -phenazine, 2- (2-hydroxy-huamazine) 2- (2, 3-hydroxy-huamadine) o2 (peroxy-huamadine) OHyW (under modified hydroxy-huamadine) imG-4-methyl-1-5-pentanoic acid 2 'amino, 2' azido, 2 'fluoro-cytidine, 2' methyl, 2 'amino, 2' azido, 2 'fluoro-adenine, 2' methyl, 2 'amino, 2' azido, 2' -fluoro-uridine, 2' -amino-2 ' -deoxyribose, 2-amino-6-chloro-purine, 2-aza-inosinyl, 2' -azido-2 ' -deoxyribose, 2' -fluoro-modified base, 2' -O-methyl-ribose, 2-oxo-7-aminopyrido-pyrimidin-3-yl, 2-oxo-pyridopyrimidin-3-yl, 2-pyridone, 3-nitropyrrole, 3- (methyl) -7- (propynyl) isoquinolonyl (carbostyrilyl), 3- (methyl) isoquinolonyl, 4- (fluoro) -6- (methyl) benzimidazole, 4- (methyl) indolyl, 4,6- (dimethyl) indolyl, 5-nitroindole, 5-substituted pyrimidine, 5- (methyl) isoquinolonyl, 5-nitroindole, 6- (aza) pyrimidine, 6- (azo), 6- (methyl) -7- (aza) indol-3-yl, 6-chloro-6-phenyl-pyrrolo-3- (3-hydroxy) -1- (aza) indole, 6- (methyl) indole, 6-chloro-6- (aza) pyrrol-3-yl, 3- (hydroxy) -2- (aza) indole ) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7- (aminoalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (aza) indol-yl, 7- (guanidyl hydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-l-yl, 7- (guanidyl hydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7- (guanidyl-hydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (guanidyl-yl Alkyl-hydroxy) -1,3- (diaza) -2- (oxo) -phenothiazin-1-yl; 7- (guanidinoalkylhydroxy) -1,3- (diaza) -2- (oxo) -phenoxazin-1-yl; 7- (propinyl) isoquinolinyl, propinyl-7- (aza) indol-3-yl, 7-deaza-inosinyl, 7-substituted 1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7-substituted 1,3- (diaza) -2- (oxo) -phenoxazin-1-yl, 9- (methyl) -imidazopyridinyl, aminoindolyl, anthracenyl, bis-ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, bis-ortho-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, difluoromethyl, inosine, imidazopyridinyl, inosinyl, isoquinolin (isoguanisine), N2-substituted purine, N6-methyl-2-amino-purine, N6-alkylated derivatives, naphthyl, nitroimidazolyl, nitroindazole, lu Bula (nu) nitro-5-hydroxy-pyrrol-2-one-3-yl O6-substituted purines; an O-alkylated derivative; ortho- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, oxo-mycin (oxoformycin) TP, para- (aminoalkylhydroxy) -6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, fused-pentaphenyl, propenthracenyl, phenyl, propynyl-7- (aza) indol-yl, pyrenyl, pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl, pyrrolo-pyrimidin-2-one-3-yl, pyrrolopyrimidinyl, pyrrolopyrazyl, stilbene (stilbenzyl), substituted 1,2, 4-triazolo-tetraphenyl, tubercidin (tubercidine), xanthine, xanthone-5' -TP, 2-thiolane, 5-aza-2-thiolane, 7-dean-2-pyrido-pyridin-3-yl, pyrrolopyrimidin-3-yl, pyrrolopyrazinyl (35) and the amino-TP forms of the amino-2-one-3-yl (pyrrolosine) TP, 2' -OH-arabino-adenosine TP, 2' -OH-arabino-cytidine TP, 2' -OH-arabino-uridine TP, 2' -OH-arabino-guanosine TP, 5- (2-methoxycarbonylvinyl) uridine TP, N6- (19-amino-pentaoxanonadecyl) adenosine TP, hydrogen (no base residues), and 2' -O-methyl-U. In some embodiments, the RNA molecule comprises a combination of at least two (e.g., 2,3, 4, or more) of the foregoing modified nucleobases. In some embodiments, 1,2, 3, 4,5 or more of the foregoing modifications may be excluded from the RNA molecules disclosed herein.

In some embodiments, the modified nucleobases in the RNA molecule comprise pseudouridine (ψ), 2-thiouridine (s 2U), 4' -thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methyluridine, 5-methoxy-uridine, 2 '-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo 5U), 5-methyl-cytidine (m 5C), a-thio-guanosine, a-thio-adenosine, 5-cyanouridine, 4' -thiouridine 7-deaza-adenine, 1-methyl-adenosine (m 1A), 2-methyl-adenine (m 2A), N6-methyl-adenosine (m 6A), 2, 6-diaminopurine, inosine (I), 1-methyl-inosine (m 1I), hui-rusoside (imG), methyl-hui-oside (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m 7G), 1-methyl-guanosine (m 1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2, 8-dimethyl adenosine, 2-geranylthiouridine, 2-lixidine, 2-selenocyanuoside, 3- (3-amino-3-carboxypropyl) -5, 6-dihydrouridine, 3- (3-amino-3-carboxypropyl) pseudouridine, 3-methyl pseudouridine, 5- (carboxyhydroxymethyl) -2' -O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenocystide, 5-aminomethyluridine, 5-carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl aminomethyl-2-geranylthiouridine, 5-carboxymethyl-aminomethyl-2-selenocystide, 5-cyanomethyluridine, 5-hydroxycytosine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-norruscogenin, 7-aminocarboxypropyl-ruscogenin methyl ester, 8-methyladenosine, N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, algardon (agmatidine), cyclic N6-threonyl carbamoyl adenosine, glutamyl-Q nucleoside, methylated under modified hydroxy huamain, N4,2' -O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Q base, preQO base, preQI base, and combinations of two or more thereof. In some embodiments, the RNA molecule includes a combination of at least two (e.g., 2, 3, 4, or more) of the foregoing modified nucleobases, including but not limited to chemical modification. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing modified nucleobases are excluded from the RNA molecules disclosed herein.

Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3C), N4-acetyl-cytidine (ac 4C), 5-formyl-cytidine (f 5C), N4-methyl-cytidine (m 4C), 5-methyl-cytidine (m 5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2C) 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, ricillin (k 2C), a-thio-cytidine, 2' -O-methyl-cytidine (Cm), 5,2' -O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2 ' -O-methyl-cytidine (ac 4 Cm), N4,2' -O-dimethyl-cytidine (m 4 Cm), 5-formyl-2 ' -O-methyl-cytidine (F5 Cm), N4,2' -O-trimethyl-cytidine (m 42 Cm), 1-thio-cytidine, 2' -F-arabino-cytidine, 2' -F-cytidine, and 2' -OH-arabino-cytidine. In some embodiments, 1,2, 3,4, 5 or more of the foregoing modified cytosines may be excluded from the RNA molecules disclosed herein.

In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides with modified uracils include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2U), 4-thio-uridine (s 4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 5-cyanouridine, 3-methyl-uridine (m 3U), 5-methoxy-uridine (mo 5U), uridine 5-oxyacetic acid (cmo 5U), and the like, Uridine 5-oxo-acetic acid methyl ester (mcmo U), 5-carboxymethyl-uridine (cm 5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm U), 5-methoxycarbonylmethyl-uridine (mcm 5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5s 2U), 5-aminomethyl-2-thio-uridine (nm 5s 2U), 5-methylaminomethyl-uridine (mcm 5U), 5-methylaminomethyl-2-thio-uridine (mcm 5s 2U), 5-methylaminomethyl-2-seleno-uridine (mcm 5se 2U), 5-carbamoylmethyl-uridine (ncm U), 5-carboxymethylaminomethyl-uridine (cmnm U), 5-carboxymethylaminomethyl-2-thiouridine (cmnmVU), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurine methyl-uridine (xm 5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouridine (xmVu), 1-taurine methyl-4-thiopseudouridine, 5-methyl-uridine (m 5U, e.g., having nucleobases deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e 1 ψ), 5-methyl-2-thiouridine (m 5s 2U), 1-methyl-4-thiopseudouridine (m 1s4 ψ), 4-thio1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5, 6-dihydrouridine, 5-methyl-dihydrouridine (m 5D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thiopseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uridine (acp 3U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp 3. Phi.), 5- (isopentenylaminomethyl) uridine (mm 5U), 5- (isopentenylaminomethyl) -2-thio-uridine (mm 5s 2U), a-thio-uridine, 2' -O-methyl-uridine (Um), 5,2' -O-dimethyl-uridine (m 5 Um), 2' -O-methyl-pseudouridine (ψm), 2-thio-2 ' -O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2 ' -O-methyl-uridine (mm 5 Um), 5-carbamoylmethyl-2 ' -O-methyl-uridine (ncm Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm Um), 3,2' -O-dimethyl-uridine (m 3 Um), and 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (mm 5 Um), 1-thiouridine, deoxythymidine, 2' -F-arabino-uridine, 2' -F-uridine, 2' -OH-arabino-uridine, 5- (2-carbonylmethoxyvinyl) uridine, and 5- [3- (l-E-propenyl) amino) ] uridine. In some embodiments, 1,2, 3,4, 5 or more of the foregoing modified uridine can be excluded from the RNA molecules disclosed herein.

In some embodiments of the invention, the modified nucleotide comprises either N1-methyl pseudouridine and/or pseudouridine.

In some embodiments, the RNA molecule comprises a nucleotide modified with N1-methyl pseudouridine. In some embodiments, the RNA molecule comprises a pseudouridine modified nucleotide.

In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the RNA molecule comprises at least one uridine N1-methyl pseudouridine substituted sequence. In some embodiments, the RNA molecule comprises a sequence in which all uridine is replaced with N1-methyl pseudouridine. N1-methyl pseudouridine is denoted "ψ" in the sequence. As used herein, the term "uracil" describes a nucleobase that can occur in a nucleic acid of an RNA. As used herein, the term "uridine" describes a nucleoside that can be present in RNA. "pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, wherein uracil is attached to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

In some embodiments, the RNA molecule comprises at least one uridine N1-methyl pseudouridine and/or pseudouridine substituted nucleic acid sequence. In some embodiments, the RNA molecule comprises at least, up to, just below, or between any two of the following (inclusive or exclusive) nucleotide sequence ：1％、2％、3％、4％、5％、6％、7％、8％、9％、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％、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% of the uridine substitution by N1-methyl pseudouridine and/or pseudouridine. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which all uridine is replaced with N1-methyl pseudouridine and/or pseudouridine.

In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases having modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m 1A), 2-methyl-adenine (m 2A), N6-methyl-adenosine (m 6A), 2-methylsulfanyl-N6-methyl-adenosine (ms 2m 6A), N6-isopentenyl-adenosine (i 6A), 2-methylsulfanyl-N6-isopentenyl-adenosine (m 6A), 2-hydroxy-5- (6-hydroxy-3-amino-adenine, 2-methyl-N6-hydroxy-5-adenine (m 6A), 2-methylsulfanyl-3-hydroxy-5-hydroxy-3-adenine (m 6A), cis-hydroxy-3-adenine (m 1A) N6-threonyl carbamoyl-adenosine (t 6A), N6-methyl-N6-threonyl carbamoyl-adenosine (m 6t 6A), 2-methylsulfanyl-N6-threonyl carbamoyl-adenosine (ms 2g 6A), N6-dimethyl-adenosine (m 62A), N6-hydroxy-N-valyl carbamoyl-adenosine (hn 6A), 2-methylsulfanyl-N6-hydroxy-N-valyl carbamoyl-adenosine (ms 2hn 6A), N6-acetyl-adenosine (ac 6A), 7-methyl-adenine, 2-methylsulfanyl-adenine, 2-methoxy-adenine, a-thio-adenosine, 2 '-O-methyl-adenosine (Am), N6,2' -O-dimethyl-adenosine (m 6 Am), N6,2 '-O-trimethyl-adenosine (m 62 Am), 1,2' -O-dimethyl-adenosine (m 1 Am), 2 '-O-ribosyl-adenosine (phosphate) (Ar (p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2' -F-arabino-adenosine, 2 '-F-adenosine, 2' -OH-arabino-adenosine, and N6- (19-amino-pentaoxanonadecyl) -adenosine in some embodiments, 1,2, 3, 4, 5 or more of the foregoing modified adenine may be excluded from the RNA molecules disclosed herein.

In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanines include inosine (I), 1-methyl-inosine (m 1I), bosyl-Q nucleoside (imG), methyl bosyl-Q nucleoside (mimG), 4-desmethyl-bosyl-guanosine (imG-14), isobornyl-guanosine (imG), bosyl-guanosine (yW), peroxy-bosyl-guanosine (o 2 yW), hydroxy-bosyl-guanosine (OhyW), under-modified hydroxy-bosyl-guanosine (OhyW), 7-deaza-guanosine, Q nucleoside (Q), epoxy-Q nucleoside (oQ), galactoside-Q nucleoside (galQ), mannosyl-Q nucleoside (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQ), gulin (G+), 7-deaza-8-aza-guanosine, 6-thio-7-deaza-guanosine (35), 6-thio-7-guanosine, 6-thio-7-aza-guanosine (6-thio-guanosine), methyl-7-guanosine (m 2), N-methyl-Q nucleoside (2), mannosyl-Q nucleoside (G2), 7-cyano-deaza-8-aza-guanosine (guanosine, 7-amino-methyl-7-dean-guanosine (15), 6-thio-guanosine (G2) and 6-thio-guanosine N2, 7-dimethyl-guanosine (m 2 '7G), N2, 7-dimethyl-guanosine (m 2' 7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thioguanosine, N2-dimethyl-6-thioguanosine, a-thioguanosine, 2 '-O-methyl-guanosine (Gm), N2-methyl-2' -O-methyl-guanosine (m 2 Gm), N2-dimethyl-2 '-O-methyl-guanosine (m 22 Gm), 1-methyl-2' -O-methyl-guanosine, N2, 7-dimethyl-2 '-O-methyl-guanosine (m 2'7 Gm), 2 '-O-methyl-inosine (Im), 1,2' -O-dimethyl-inosine (m 1), 2 '-O-methyl-guanosine (Im), 2' -O-methyl-guanosine (m), and guanosine (p-methyl-guanosine). In some embodiments, 1,2, 3, 4, 5 or more of the foregoing modified guanines may be excluded from the RNA molecules disclosed herein.

In some embodiments, for a particular modification, the RNA molecule is uniformly modified (e.g., fully modified, modified throughout the sequence). In some embodiments, the RNA molecule may be partially or completely (e.g., uniformly) modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., any or more or all of purine and/or pyrimidine, or A, G, U, C) in a polynucleotide of the invention or a given predetermined sequence region thereof may be uniformly modified. In some embodiments, all nucleotides X in a polynucleotide of the invention (or in a given sequence region thereof) are modified nucleotides, wherein X can be any of nucleotides A, G, U, C, and/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 and/or a+g+c. For example, the polynucleotide may be uniformly modified with pseudouridine, meaning that all uridine residues in the RNA sequence are replaced with pseudouridine. Similarly, polynucleotides may be uniformly modified for any type of nucleoside residue present in a sequence by substitution with modified residues such as those set forth above. The modified nucleotide may be replaced by one 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).

The RNA molecule may contain modified nucleotides (related to the overall nucleotide content, or to one or more types of nucleotides (e.g., any one or more of A, G, U and/or C)) at or about 1% -100% (e.g., at least, up to, just below, or between any two of the following (inclusive or exclusive )：1、2、3、4、5、6、7、8、9、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、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、99 or 100%) or any intermediate percentage (e.g., 1％～20％、1％～25％、1％～50％、1％～60％、1％～70％、1％～80％、1％～90％、1％～95％、10％～20％、10％～25％、10％～50％、10％～60％、10％～70％、10％～80％、10％～90％、10％～95％、10％～100％、20％～25％、20％～50％、20％～60％、20％～70％、20％～80％、20％～90％、20％～95％、20％～100％、50％～60％、50％～70％、50％～80％、50％～90％、50％～95％、50％～100％、70％～80％、70％～90％、70％～95％、70％～100％、80％～90％、80％～95％、80％～100％、90％～95％、90％～100％、 and 95% -100%). It is understood that any remaining percentages are complemented by the presence of unmodified A, G, U and/or C.

In some embodiments, the RNA molecule can include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.

In some embodiments, the RNA molecule can include one or more structural and/or chemical modifications and/or alterations that confer upon the polynucleotide suitable properties, in some embodiments, reduced degradation in a cell or organism, and/or substantial induction of an innate immune response in a cell into which the RNA molecule is introduced. As used herein, a "structural" feature or modification is a feature or modification in which two or more linked nucleotides are inserted, deleted, duplicated, inverted, and/or randomized in an RNA molecule without significantly chemically modifying the nucleotides themselves. The structural modification is of a chemical nature and is therefore a chemical modification, as the chemical bond will necessarily break and reform to effect the structural modification. However, structural modifications will result in different nucleotide sequences. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be modified from the "ATCG" structure to "ATCCCG". Here, a dinucleotide "CC" has been inserted, resulting in structural modification of the polynucleotide.

In some embodiments, the modified RNA molecule introduced into the cell or organism exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. In some embodiments, a modified RNA molecule introduced into a cell or organism may exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, the RNA molecule can include one or more modified nucleotides in addition to any 5' cap structure. In some embodiments, the RNA molecule does not include modified nucleotides, e.g., modified nucleobases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G and C, which are described further below, except for an optionally 5' cap that may include, e.g., 7-methylguanosine. In some embodiments, the RNA can include a 5 'cap comprising 7' -methylguanosine, and the first 1,2, or 35 'ribonucleotides can be methylated at the 2' position of the ribose.

[ B.5' cap ]

In some embodiments, the RNA molecules described herein include a 5 'cap, which generally "caps" the 5' end of the RNA and stabilizes the RNA molecule.

In some embodiments, the 5 'cap portion is a natural 5' cap. "native 5 'cap" is defined to include a cap of 7-methylguanosine bonded to the 5' end of the mRNA molecule via a 5'-5' triphosphate linkage. In some embodiments, guanosine included in the 5' cap may be modified, for example, by methylation at one or more positions (e.g., at position 7) on the base (guanine) and/or by methylation at one or more positions of ribose. In some embodiments, the guanosine nucleoside included in the 5' cap comprises a 3' o methylation at ribose (3 ' ome g). In some embodiments, the guanosine included in the 5' cap comprises methylation at the 7 position of guanine (m 7G). In some embodiments, the guanosine nucleoside included in the 5' cap comprises methylation at the 7 position of guanine and 3' o methylation at ribose (m 7 (3 ' ome)). The 5' cap may be incorporated during RNA synthesis (e.g., co-transcription capping), or may be enzymatically engineered after RNA transcription (e.g., post-transcription capping). In some embodiments, co-transcription capping with the caps disclosed herein increases the capping efficiency of RNA compared to co-transcription capping with an appropriate reference comparator. In some embodiments, increasing capping efficiency may increase the translation efficiency and/or translation rate of the RNA, and/or increase expression of the encoded polypeptide. In some embodiments, capping is performed after purification of the RNA molecule (e.g., tangential flow filtration).

In some embodiments, the RNAs described herein comprise a 5 'cap or 5' cap analog, such as cap 0, cap 1, or cap 2. In some embodiments, the provided RNAs do not have uncapped 5' -triphosphates. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide. In some embodiments, the 5 'cap portion is a 5' cap analogue. In some embodiments, the RNA can be capped with a 5' cap analog. Cap structures include, but are not limited to ⁷mG(5')ppp(5')N₁pN₂ p (cap 0), ⁷mG(5')ppp(5')N₁ ^m pNp (cap 1), and ⁷mG(5')ppp(5′)N₁ ^mpN₂ ^m p (cap 2). In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing cap structures may be excluded from the RNA molecules disclosed herein.

In some embodiments, the RNA described herein comprises cap 0. In some embodiments, cap 0 is N7-methylguanosine and the cap 0 structure comprises guanosine (m 7G) methylated at the 7 position of guanine. In some embodiments, the cap 0 structure is linked to RNA via a 5'-5' triphosphate linkage, and is also referred to herein as m7G, m7Gppp and/or m7G (5 ') ppp (5'). The 5 'cap may be methylated with structure ⁷mG(5′)ppp(5′)N₁pN₂ p (cap 0) or a derivative thereof, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5 'cap, typically the 5' end of the mRNA. An exemplary enzymatic reaction for capping may include the use of vaccinia Virus Capping Enzymes (VCEs) including mRNA triphosphatase, guanylate transferase, and guanine-7-methyltransferase, which catalyze the construction of the N7-monomethylated cap 0 structure. The cap 0 structure plays an important role in maintaining the stability and translational efficacy of RNA molecules. In cells, the cap 0 structure is critical for efficient translation of cap-carrying mRNA.

In some embodiments, the RNA described herein comprises cap 1, e.g., as described herein. The 5 'cap of the RNA molecule may be further modified at the 2' O position by a2 '-O-methyltransferase which causes the production of cap 1 structure (m 7Gppp [ m2' -O ] N), which may further increase translation efficiency. In some embodiments, the cap 1 structure comprises guanosine (m 7G) methylated at the 7 position of guanine and the first nucleotide (2 'ome n ₁) methylated by 2' o in RNA. in some embodiments, cap 1 structure is linked to RNA via a 5'-5' triphosphate linkage and is also referred to herein as m7GpppN ^m, where N ^m represents any nucleotide with 2'o methylation, ⁷mG(5')ppp(5′)N₁ ^mpNp、m7Gppp(2′OMeN₁) and/or m7G (5') ppp (5 ') (2' ome N ₁). In some embodiments, N ₁ is selected from A, C, G or U. In some embodiments, N ₁ is a. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U. In some embodiments, the m7G (5 ') ppp (5') (2 'ome N ₁) cap 1 structure comprises a second nucleotide N ₂, which is a cap proximal nucleotide at position 2 and is selected from A, G, C or U (m 7G (5') ppp (5 ') (2' ome N ₁)N₂). In some embodiments, N ₂ is a. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U.

In some embodiments, the cap 1 structure comprises guanosine (m 7G) methylated at the 7 position of guanine and one or more additional modifications (e.g., methylation on ribose) and a first nucleotide in RNA that is methylated by 2' o. In some embodiments, the cap 1 structure comprises guanosine methylated at the 7 position of guanine, 3'o methylation at ribose (m 7 (3' ome)) and a first nucleotide in RNA that is methylated with 2'o (2' ome n ₁). In some embodiments, cap 1 structure is linked to RNA via a 5'-5' triphosphate linkage, and is also referred to herein as m7 (3 'ome g) ppp (2' ome n ₁) and/or m7 (3 'ome g) (5') ppp (5 ') (2' ome n ₁). In some embodiments, N ₁ is selected from A, C, G or U. In some embodiments, N ₁ is a. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U. In some embodiments, the m7 (3 'ome g) (5') ppp (5 ') (2' ome N ₁) cap 1 structure comprises a second nucleotide N ₂ that is a cap proximal nucleotide at position 2 and is selected from A, G, C or U (m 7 (3 'ome g) (5') ppp (5 ') (2' ome N ₁)N₂). In some embodiments, N ₂ is a. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U. in some embodiments, 1,2, 3,4, 5 or more of the foregoing cap 1 structures may be excluded from the RNA molecules disclosed herein.

In some embodiments, the second nucleotide in the cap 1 structure may comprise one or more modifications, such as methylation. In some embodiments, the RNA described herein comprises cap 2. In some embodiments, the cap 1 structure comprising the second nucleotide comprising 2' o methylation is a cap 2 structure.

In some embodiments, the RNA molecule can be enzymatically capped at the 5' end using vaccinia guanylate transferase, guanylate, and S-adenosyl-L-methionine, resulting in a cap 0 structure. An inverted 7-methylguanosine cap is added via a 5'-5' triphosphate bridge. Alternatively, the cap 1 structure is obtained using a2 'O-methyltransferase with vaccinia guanylate transferase, wherein the 2' OH group on the penultimate nucleotide is methylated in addition to the cap 0 structure. S-adenosyl-L-methionine (SAM) is a cofactor for the use as a methyltransferase. Non-limiting examples of 5' cap structures are those structures that have, inter alia, increased binding, increased half-life, reduced sensitivity to 5' endonucleases, and/or reduced 5' uncapping of cap binding polypeptides as compared to synthetic 5' cap structures (or wild-type, natural or physiological 5' cap structures) known in the art.

For example, recombinant vaccinia virus capping enzymes and recombinant 2 'O-methyltransferases can create a typical 5' -5 'triphosphate linkage between the 5' terminal nucleotide of the mRNA and the guanine cap nucleotide, where the cap guanine comprises N7 methylation and the 5 'terminal nucleotide of the mRNA comprises a 2' -O-methyl group. Such a structure is referred to as a cap 1 structure. This cap results in a higher translational capacity and cell stability and reduced activation of the cell pro-inflammatory cytokines compared to, for example, other 5' cap analogue structures known in the art.

The cap species may include one or more modified nucleosides and/or linker moieties. For example, the cap may include a guanine nucleotide joined at its 5' position by a triphosphate linkage and a guanine (G) nucleotide methylated at the 7 position, e.g., m7G (5 ') ppp (5 ') G, typically written as m7GpppG. The cap species may also be an anti-inversion cap analogue. A non-limiting list of possible cap species includes m7GpppG、m7Gpppm7G、m73'dGpppG、m27,O3'GpppG、m27,O3′GppppG、m27,O2′GppppG、m7Gpppm7G、m73′dGpppG、m27,O3′GpppG、m27,O3'GppppG and m27, O2' GppppG. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing cap species may be excluded from the RNA molecules disclosed herein.

In some embodiments, the 5 'end cap comprises a cap analog, e.g., the 5' end cap may comprise a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2' -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, 1,2,3, 4, 5 or more of the foregoing guanine analogs can be excluded from the cap structures disclosed herein.

In some embodiments, the capping region may comprise a single cap or a series of nucleotides forming a cap. In this embodiment, the capping region may be 1 to 10, for example 2 to 9,3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2 or 10 or fewer nucleotides in length. In this embodiment, the capping region is at least, up to, just below, or between any two of (inclusive or exclusive) 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, no cap is present. In some embodiments, the first and second operable regions may be in the range of 3-40 nucleotides in length, e.g., 5-30, 10-20, 15, or at least 4 or 30 or less, and may include one or more signal and/or restriction sequences in addition to the start and/or stop codons. In some embodiments, the first and second manipulation regions are at least, up to, just below, or between any two of the following (including or exclusive )：3、4、5、6、7、8、9、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 or 40 nucleotides, and may include one or more signal and/or restriction sequences in addition to the start and/or stop codons).

Other examples of 5 'cap structures include, but are not limited to, glyceryl, inverted deoxyabasic residues (moieties), 4',5 '-methylene nucleotides, 1- (. Beta. -D-erythrofuranosyl) nucleotides, 4' -thio nucleotides, carbocyclic nucleotides, 1, 5-anhydrous hexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4' -open-loop nucleotides, acyclic 3, 4-dihydroxybutyl nucleotides, acyclic 3, 5-dihydroxypentyl nucleotides, 3'-3' -inverted nucleotide moieties, 3'-3' -inverted abasic moieties, 3'-2' -inverted nucleotide moieties, 3'-2' -inverted abasic moieties, 1, 4-butanediol phosphates, 3 '-phosphoramidates, hexyl phosphates, aminohexyl phosphates, 3' -phosphorothioates, phosphorodithioates, and/or bridged or non-bridged methylphosphonate moieties. In some embodiments, 1,2, 3,4, 5 or more of the foregoing 5' cap structures may be excluded from the RNA molecules disclosed herein.

In some embodiments, the RNA molecules of the invention comprise at least one 5' cap structure. In some embodiments, the RNA molecules of the invention do not comprise a 5' cap structure.

A variety of synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska,E.,Kowalska,J.,Su,W.,Kuhn,A.N.,Slepenkov,S.V.,Darynkiewicz,E.,Sahin,U.,Jemielity,J. and Rhoads,R.E.,Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology 69(Rabinovich,P.H., code), 2013. In one embodiment, the 5' capping structure comprises a modified 5' cap 1 structure (m ⁷G⁺ m3' -5' -ppp-5' -Am). In one embodiment, the 5 'capping structure comprises (3' ome) -m ₂ ⁷,3′^-OGppp(m₁ ^2′-O) ApG (TriLink BioTechnologies). This molecule is identical to the native RNA cap structure in that it starts with guanosine methylated at N7 and is bound by a 5'-5' triphosphate linkage to the first encoded nucleotide of the transcribed RNA (in this case adenosine). This guanosine is also methylated at the 3' hydroxyl group of the ribose to alleviate possible reverse incorporation of the cap molecule. The 2' hydroxyl group of ribose on adenosine is methylated, giving the cap 1 structure.

[ C. untranslated region (UTR) ]

The 5'utr is a regulatory region located at the 5' end of the open reading frame of a protein that is transcribed into mRNA but is not translated into an amino acid sequence, and/or a corresponding region in an RNA polynucleotide (such as an mRNA molecule). The untranslated region (UTR) may be present at the 5 'end (upstream) (5' UTR) of the open reading frame and/or the 3 'end (downstream) (3' UTR) of the open reading frame.

In some embodiments, the UTR is derived from mRNA that is naturally abundant in the particular tissue (e.g., lymphoid tissue) to which mRNA expression is targeted. In some embodiments, UTR increases protein synthesis. Without being bound by a mechanism or theory, UTR may increase protein synthesis by increasing the retention time of mRNA in the translated polysomes (message stability) and/or the rate at which the ribosome initiates translation based on the message (message translation efficiency). Thus, UTR sequences can prolong protein synthesis in a tissue-specific manner.

In some embodiments, regulatory features of UTRs may be incorporated into the RNAs of the present invention to, inter alia, enhance stability of the molecule. Specific features may also be incorporated to ensure controlled down-regulation of transcripts in cases where they are misdirected to undesired organ sites. Various 5'UTR and 3' UTR sequences are known and available in the art.

It is understood that any UTR from any gene may be incorporated into a region of RNA of the present invention. In addition, a variety of wild-type UTRs of any known genes may be utilized. It is also within the scope of the invention to provide artificial UTRs that are not variants of the wild-type region. The placement orientation of these UTRs or portions thereof may be the same as in the transcripts from which they are selected, or the orientation and/or position may vary. Thus, the 5 'and/or 3' utrs may be inverted, shortened, lengthened, and/or one or more other 5 'utrs or 3' utrs as well. As used herein, the term "altering" when related to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. For example, the 5'UTR and/or 3' UTR may be altered relative to a wild-type or natural UTR by altering its orientation and/or position as taught above, and/or may be altered by including additional nucleotides, deleted nucleotides, exchanged and/or translocated nucleotides. Any of these changes produces "altered" UTRs (whether 5 'and/or 3'), including variant UTRs.

In some embodiments, dual, triple, or quadruple UTRs, such as 5 'and/or 3' UTRs, may be used. As used herein, a "dual" UTR is a UTR in which two copies of the same UTR are serially or substantially serially encoded. For example, a dual beta-globulin 3' UTR may be used. It is also within the scope of the invention to have a patterned UTR. As used herein, "patterned UTRs" are those UTRs reflecting a repeating or alternating pattern, such as AB or AABBAABBAABB or abcapcabc, or variants thereof, repeated once, twice, or more than 3 times. In these modes, each letter A, B or C represents a different UTR at the nucleotide level.

The RNA may encode a polypeptide of interest belonging to a family of proteins expressed in a particular cell, tissue, and/or at some time during development. In some embodiments, UTRs from any of these genes may exchange any other UTRs of the same or different protein family to create new RNA molecules. 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, and/or expression pattern.

In some embodiments, the 5'utr and 3' utr sequences are calculated. In some embodiments, the 5'utr and the 3' utr are derived from mRNA naturally abundant in tissue. The tissue may be, for example, liver, stem cells and/or lymphoid tissue. Lymphoid tissues may include, for example, any of lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and/or natural killer cells), macrophages, mononuclear spheres, dendritic cells, neutrophils, eosinophils, and reticulocytes. In some embodiments, the 5'utr and the 3' utr are derived from an alphavirus (alphavirus). In some embodiments, the 5'utr and the 3' utr are from wild-type alphaviruses.

In some embodiments, the untranslated region may also include a translation enhancer component (TEE). As a non-limiting example, TEEs may include those described in U.S. application No. 20090226470 (which is incorporated herein by reference in its entirety) as well as those known in the art.

【i.5'UTR】

In some embodiments, the RNAs disclosed herein comprise a 5' utr. The 5'UTR, if present, is located at the 5' end and begins at a transcription start site upstream of the start codon of the protein coding region. The 5' utr is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. The 5'utr may contain various regulatory components, such as a 5' cap structure, a stem-loop structure, and an Internal Ribosome Entry Site (IRES), which may play a role in the control of translation initiation. The 5' UTR may contain markers, such as Kozak sequences, which also involve ribosomes to initiate many gene translation processes. The 5' UTR may also form secondary structures that involve elongation factor binding.

In some embodiments, the 5' utrs disclosed herein comprise, for example, cap proximal sequences disclosed herein. In some embodiments, the cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, the cap proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, the cap structure comprises one or more polynucleotides of the cap proximal sequence. In some embodiments, the cap structure comprises nucleotide +1 (N ₁) of the m7 guanosine cap and the RNA polynucleotide. In some embodiments, the cap structure comprises nucleotide +2 (N ₂) of the m7 guanosine cap and the RNA polynucleotide. In some embodiments, the cap structure comprises nucleotides +1 and +2 (N ₁ and N ₂) of the m7 guanosine cap and the RNA polynucleotide.

Those skilled in the art who review the present invention will appreciate that in some embodiments, one or more residues of the cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in the RNA by way of inclusion in the cap entity (e.g., cap 1 structure, etc.), or that in some embodiments, at least some of the residues in the cap proximal sequence may be added enzymatically (e.g., by a polymerase, such as a T7 polymerase). For example, in certain exemplary embodiments in which a (m ₂ ^7,3'-O)Gppp(m^2'-O) ApG cap is utilized, the +1 and +2 residues are capped (m ₂ ^7,3'-O) a and G residues, and the +3, +4, and +5 residues are added by a polymerase (e.g., T7 polymerase).

In some embodiments, the cap proximal sequence comprises N ₁ and/or N ₂ of the cap structure, wherein N ₁ and N ₂ are any nucleotide, such as A, C. G or U. In some embodiments, N ₁ is a. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U. In some embodiments, N ₂ is a. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U. In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ and N ₃、N₄ and N ₅ of the cap structure, wherein N ₁～N₅ corresponds to position +1 of the RNA polynucleotide, +2, +3, +4 and/or +5. In some embodiments, N ₁、N₂、N₃、N₄ or N ₅ is any nucleotide, e.g., A, C, G or U. In some embodiments, N ₁N₂ comprises any of AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG or UU. In some implementations, N ₁N₂ includes AG and N ₃N₄N₅ includes any one of ：AAA、ACA、AGA、AUA、AAG、AGG、ACG、AUG、AAC、ACC、AGC、AUC、AAU、ACU、AGU、AUU、CAA、CCA、CGA、CUA、CAG、CGG、CCG、CUG、CAC、CCC、CGC、CUC、CAU、CCU、CGU、CUU、GAA、GCA、GGA、GUA、GAG、GGG、GCG、GUG、GAC、GCC、GGC、GUC、GAU、GCU、GGU、GUU、UAA、UCA、UGA、UUA、UAG、UGG、UCG、UUG、UAC、UCC、UGC、UUC、UAU、UCU、UGU or UUU of the following.

In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ of the cap structure, and a sequence comprising A ₃A₄X₅ (SEQ ID NO:46; wherein X ₅ is A, G, C or U), wherein N ₁ and N ₂ are each independently selected from A, C, G or U. In some embodiments, N ₁ is a and N ₂ is G. In some embodiments, X ₅ is selected from A, C, G or U. In some embodiments, X ₅ is a. In some embodiments, X ₅ is C. In some embodiments, X ₅ is G. In some embodiments, X ₅ is U.

In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ of the cap structure, and a sequence comprising C ₃A₄X₅ (SEQ ID NO:47; wherein X ₅ is A, G, C or U), wherein N ₁ and N ₂ are each independently selected from A, C, G or U. In some embodiments, N ₁ is a and N ₂ is G. In some embodiments, X ₅ is selected from A, C, G or U. In some embodiments, X ₅ is a. In some embodiments, X ₅ is C. In some embodiments, X ₅ is G. In some embodiments, X ₅ is U.

In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ of the cap structure, and comprises X ₃Y₄X₅ (SEQ ID NO:48; wherein X ₃ or X ₅ are each independently selected from A, G. C or U, and Y ₄ is not a sequence of C). In some embodiments, N ₁ and N ₂ are each independently selected from A, C, G or U. In some embodiments, N ₁ is a and N ₂ is G. In some embodiments, X ₃ and X ₅ are each independently selected from A, C, G or U. In some embodiments, X ₃ and/or X ₅ are a. in some embodiments, X ₃ and/or X ₅ are C. In some embodiments, X ₃ and/or X ₅ are G. In some embodiments, X ₃ and/or X ₅ are U. In some embodiments, Y ₄ is C. In other embodiments, Y ₄ is not C. In some embodiments, Y ₄ is a. In some embodiments, Y ₄ is G. In other embodiments, Y ₄ is not G. In some embodiments, Y ₄ is U.

In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ of the cap structure and a sequence comprising A ₃C₄A₅ (SEQ ID NO: 49). In some embodiments, N ₁ and N ₂ are each independently selected from A, C, G or U. In some embodiments, N ₁ is a and N ₂ is G.

In some embodiments, the cap proximal sequence comprises N ₁ and N ₂ of the cap structure and a sequence comprising A ₃U₄G₅ (SEQ ID NO: 50). In some embodiments, N ₁ and N ₂ are each independently selected from A, C, G or U. In some embodiments, N ₁ is a and N ₂ is G.

In some embodiments, 1, 2, 3,4, 5 or more of the foregoing cap proximal sequences may be excluded from the 5' utr of the RNA molecules disclosed herein.

In some embodiments of the invention, the 5' UTR is a heterologous UTR, e.g., a UTR found in nature to bind to a different ORF. In another embodiment, the 5' UTR is a synthetic UTR that does not exist in nature, for example. Synthetic UTRs include UTRs that have been mutated or synthesized to improve their properties (e.g., to increase gene expression). In some embodiments, the 5' utr is functionally linked to, e.g., binds to, the ORF such that it can function, e.g., increase, enhance, stabilize, and/or prolong protein production from the RNA molecule, and/or increase protein expression and/or total protein production from the RNA molecule, as compared to a reference RNA molecule comprising a reference 5' utr or an RNA molecule lacking the 5' utr. In some embodiments, 1, 2, 3, 4, 5, or more of the aforementioned 5' utr functions may be excluded.

Exemplary 5' UTRs include 5' UTRs derived from Xenopus (Xenopus) or human alpha or beta globulin, human cytochrome b-245a, hydroxysteroid (17 b) dehydrogenase, tobacco etch virus, CMV immediate early 1 (IE 1) gene, TEV, HSP705', c-Jun, or homologs, fragments, or variants of any of the foregoing. In some embodiments, the 5'UTR is a fragment, homolog or variant of the 5' UTR of the TOP gene that lacks the 5'TOP motif (oligo-pyrimidine tract), the 5' UTR derived from the ribosomal protein large 32 (L32) gene, the 5'UTR derived from the 5' UTR of the hydroxysteroid (17 p) dehydrogenase 4 gene (HSD 17B 4), or the 5'UTR derived from the 5' UTR of ATP5A 1. In some embodiments, the 5' UTR is derived from patent application WO2013/143700 (the disclosure of which is incorporated herein by reference in its entirety) from SEQ ID NO: 1-1363, SEQ ID NO:1395, SEQ ID NO:1421, and SEQ ID NO:1422, or sequences having at least, up to, just below, or between any two of the foregoing (inclusive or exclusive) identity to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. Sequence GGGAUCCUACC may also be used. In some embodiments, 1,2, 3,4,5 or more of the foregoing 5' utr sequences may be excluded from the RNA molecules disclosed herein.

In some embodiments, the 5'utr comprises a sequence ：RPSA、RPS2、RPS3、RPS3A、RPS4、RPS5、RPS6、RPS7、RPS8、RPS9、RPS10、RPS1 1、RPS12、RPS13、RPS14、RPS15、RPS15A、RPS16、RPS17、RPS18、RPS19、RPS20、RPS21、RPS23、RPS24、RPS25、RPS26、RPS27、RPS27A、RPS28、RPS29、RPS30、RPL3、RPL4、RPL5、RPL6、RPL7、RPL7A、RPL8、RPL9、RPL10、RPL10A、RPL1 1、RPL12、RPL13、RPL13A、RPL14、RPL15、RPL17、RPL18、RPL18A、RPL19、RPL21、RPL22、RPL23、RPL23A、RPL24、RPL26、RPL27、RPL27A、RPL28、RPL29、RPL30、RPL31、RPL32、RPL34、RPL35、RPL35A、RPL36、RPL36A、RPL37、RPL37A、RPL38、RPL39、RPL40、RPL41、RPLPO、RPLP1、RPLP2、RPLP3、RPLPO、RPLP1、RPLP2、EEF1A1、EEF1 B2、EEF1 D、EEF1 G、EEF2、EIF3E、EIF3F、EIF3H、EIF2S3、EIF3C、EIF3K、EIF3EIP、EIF4A2、PABPC1、HNRNPA1、TPT1、TUBB1、UBA52、NPM1、ATP5G2、GNB2L1、NME2、UQCRB; from the 5' utr region of a gene encoding each or from a homolog, fragment or variant thereof or a gene sequence having at least, up to, just below, or between any two of (inclusive or exclusive) 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to any of the foregoing gene sequences. In some embodiments, 1,2, 3,4, 5 or more of the foregoing 5' utr sequences may be excluded from the RNA molecules disclosed herein.

In one embodiment, the DNA encoding the 5' UTRs disclosed herein comprises a sequence 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to, at least, up to, just below, or between any two of (inclusive or exclusive of) SEQ ID NO 17. In one embodiment, the DNA encoding the 5' UTR comprises the sequence of SEQ ID NO: 17. In one embodiment, the RNAs disclosed herein comprise 5' utrs comprising sequences having at least, up to, just below, or between any two of the following, identity to the 5' utrs provided in either of SEQ ID NOs 18 or 19 (wherein the transcribed 5' cap structures are underlined) 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one embodiment, the 5'UTR comprises the sequence of either of SEQ ID NO:18 or 19 (wherein the transcribed 5' cap structure is underlined).

【SEQ ID NO:17(DNA)】

AGAATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCC

【SEQ ID NO:18(RNA)】

AGAAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCC

【SEQ ID NO:19(RNA)】

AGAAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCAC AGACΨCAGAG AGAACCC

In one embodiment, the DNA encoding the 5' UTRs disclosed herein comprises a sequence 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to, at least, up to, just below, or between any two of (inclusive or exclusive of) SEQ ID NO. 51. In one embodiment, the DNA encoding the 5' UTR comprises the sequence of SEQ ID NO: 51. In one embodiment, the RNAs disclosed herein comprise 5 'utrs comprising sequences having at least, up to, just below, or between any two of the following, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the 5' utrs provided in any one of SEQ ID NOs 52 or 53. In one embodiment, the 5'UTR comprises the sequence of either of SEQ ID NO:52 or 53 (wherein the transcribed 5' cap structure is underlined).

【SEQ ID NO:51(DNA)】

GATAGGCGGC GCATGAGAGA AGCCCAGACC AATTACCTAC CCAAA

【SEQ ID NO:52(RNA)】

GAUAGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA

【SEQ ID NO:53(RNA)】

GAΨAGGCGGC GCAΨGAGAGA AGCCCAGACC AAΨΨACCΨAC CCAAA

In some embodiments, 1, 2, 3, or more of the foregoing 5' utr sequences may be excluded from the RNA molecules disclosed herein.

【ii.3′UTR】

In some embodiments, the RNAs disclosed herein comprise a 3' utr. The 3' UTR, if present, is located downstream of the open reading frame of the protein coding sequence, e.g., downstream of the stop codon of the protein coding region. The 3' UTR is typically the portion of mRNA located between the protein coding sequence of the mRNA and the poly-A tail. Thus, in some embodiments, the 3' utr is upstream of (if present) the poly a sequence, e.g., immediately adjacent to the poly a sequence. The 3' utr may be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

The natural or wild-type 3' UTR comprises an extension of adenosine and uridine. These AU-rich markers are particularly prevalent in genes with higher turnover rates. Based on their sequence characteristics and functional properties, AU-rich components (AREs) can be divided into three classes, class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. Class II AREs have two or more overlapping UUAUUUA (U/A) (U/A) nonamers. Class III ARE free of the AUUUA motif. Most proteins that bind ARE known to destabilize the molecule. Thus, the introduction, removal and/or modification of 3' utrare may be used to modulate the stability of a nucleic acid (e.g., RNA) of the present invention. When engineering a particular nucleic acid, in some embodiments, one or more ARE copies can be introduced to make the RNA less stable and thus reduce translation and reduce the production of the resulting protein. Also, in some embodiments, AREs can be identified and removed and/or mutated to increase intracellular stability and thus increase translation and production of the resulting protein. Transfection experiments can be performed using the nucleic acids of the invention in relevant cell lines, and protein production can be analyzed at various time points after transfection. For example, the molecules can be engineered with different ARE and the resulting proteins analyzed by transfecting cells using ELISA kits for the relevant proteins and 6 hours, 12 hours, 24 hours, 48 hours, and 7 days post-transfection. In some embodiments, the 3' utr may allow one or more AU-rich sequences to be removed. Alternatively the AU-rich sequence may remain in the 3' utr.

The 3' utr may also comprise components that are not encoded in the template of transcribed RNA but that are added during maturation after transcription, e.g. a poly a tail. The 3' UTR of mRNA is not translated into amino acid sequences. In some embodiments, the RNAs disclosed herein comprise a 3' utr comprising an F component and/or an I component. In some embodiments, the 3' utr or proximal sequence thereof comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an Xhol site.

In some embodiments of the invention, the 3' UTR is a heterologous UTR, e.g., a UTR found in nature to bind to a different ORF. In another embodiment, the 3' UTR is a synthetic UTR that does not exist in nature, for example. In some embodiments, the 3' utr is functionally linked to, e.g., binds to, the ORF such that it can function, e.g., increase, enhance, stabilize, and/or prolong protein production from the RNA molecule, and/or increase protein expression and/or total protein production from the RNA molecule, as compared to a reference RNA molecule comprising a reference 3' utr or an RNA molecule lacking the 3' utr. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing 3' utr functions may be excluded.

Exemplary 3' UTRs include those derived from the 3' UTR of albumin genes, alpha-globulin genes, beta-globulin genes, ribosomal protein genes, tyrosine hydroxylase genes, lipoxygenase genes, and collagen alpha genes (such as collagen alpha 1 (1) genes), or from homologues, fragments or variants of the 3' UTR comprising the genes of albumin genes, alpha-globulin genes, beta-globulin genes, ribosomal protein genes, tyrosine hydroxylase genes, lipoxygenase genes, and/or collagen alpha genes (such as collagen alpha 1 (1) genes), sequences having identity of at least, up to, just below, or between any two of the foregoing sequences (including or exclusive) 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% in some embodiments of the sequences according to SEQ ID NO: 1369-1390 of patent application WO2013/143700, the disclosure of which is incorporated herein by reference in its entirety, or sequences of any two of the foregoing, in other embodiments of the invention, UU 3, UU 2, or more herein disclosed.

In some embodiments, the 3' UTR comprises a sequence of transcripts that includes NM_000661.4、NM_001024921.2、NM_000967.3、NM_001033853.1、NMJD00968.3、NM_000969.3、NM_001024662.1、NM_000970.3、NM_000971.3、NMJD00972.2、NM_000975.3、NM_001 199802.1、NM_000976.3、NM__000977.3、NM_033251.2、NMJ 01243130.1、NM_001243131、NM_000978.3、NM_000979.3、NM_001270490.1、NMJD00980.3、NM_000981.3、NM_000982.3、NM_000983.3、NM_000984.5、NM_000985.4、NM_001035006.2、NM_001 199340.1、NM_001 199341.1、NMJD01 199342.1、NM_001199343.1、NM_001 199344.1、NM_001 199345.1、NM_000986.3、NM_000987.3、NM_000988.3、NM_000989.3、NM_000990.4、NM_001 136134.1、NMJD00991.4、NM_001 136135.1、NM_001 136136.1、NM_001 136137.1、NM_000992.2、NM_000993.4、NM_001098577.2、NM_001099693.1、NM_000994.3、NM_001007073.1、NM_001007074.1、NM_000996.2、M_000997.4、NM_000998.4、NM_000999.3、NM_001035258.1、NM_001000.3、NM_001002.3、NM_053275.3、NM_001003.2、NM_213725.1、NM_001004.3、NM_001005.4、NM_001256802.1、NM_001260506.1、NM_001260507.1、NM_001006.4、NM_001267699.1、NM_001007.4、NM_001008.3、N_001009.3、NM_001010.2、NM_00101 1.3、NM_001012.1、NM_001013.3、NM_001203245.2、NM_001014.4、NM_001204091.1、NM_001015.4、NM_001016.3、NM_001017.2、NM_001018.3、NM_001030009.1、NM_001019.4、NM_001020.4、NM_001022.3、NM_001 146227.1、NM_001023.3、NM_001024.3、NM_001025.4、NM_001028.2、NM_001029.3、NM_001030.4、NM_002954、NM_001 135592.2、NM_001 177413.1、NM_001031.4、NM_001032.4、NM_001030001.2、NM_002948.3、NM_001253379.1、NM_001253380.1、NM_001253382.1、NM_001253383.1、NM_001253384.1、NM_002952.3、NM_001034996.2、NM_001025071.1、NM_001025070.1、NM_005617.3、NM_006013.3、NM_001256577.1、NM_001256580.1、NM_007104.4、NM_007209.3、NM_012423.3、NM_001270491.1、NM_033643.2、NM_015414.3、NM_021029.5、NM_001 199972.1、NM_021 104.1、NM_022551.2、NM_033022.3、NM_001 142284.1、NM_001026.4、NM_001 142285.1、NM_001 142283.1、NM_001 142282.1、NM_000973.3、NM_033301.1、NM_000995.3、NM_033625.2、NM_001021.3、NM_002295.4、NM_001012321.1、NM_001033930.1、NM_003333.3、NM_001997.4、NM_001099645.1、NM_001021.3、NM_052969.1、NM_080746.2、NM_001001.4、NM_005061.2、NM_015920.3、NM_016093.2、NM_198486.2、NG_01 1 172.1、NG_011253.1、NG_000952.4、NR_002309.1、NG_010827.2、NG_009952.2 or NG_009517.1, or a sequence of transcripts that have at least, up to, just below, or between any two of the foregoing (inclusive or exclusive) identity of 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing 3' utr sequences may be excluded from the RNA molecules disclosed herein.

In some embodiments, the 3'UTR comprises a sequence from the 3' UTR region of a gene encoding a ribosomal protein, e.g., ribosomal protein L9 (RPL 9), ribosomal protein L3 (RPL 3), ribosomal protein L4 (RPL 4), ribosomal protein L5 (RPL 5), ribosomal protein L6 (RPL 6), ribosomal protein L7 (RPL 7), ribosomal protein L7A (RPL 7A), ribosomal protein L11 (RPL 11), ribosomal protein L12 (RPL 12), ribosomal protein L13 (RPL 13), ribosomal protein L23 (RPL 23), ribosomal protein L5 (RPL 23), Ribosomal protein L18 (RPL 18), ribosomal protein L18A (RPL 18A), ribosomal protein L19 (RPL 19), ribosomal protein L21 (RPL 21), ribosomal protein L22 (RPL 22), ribosomal protein L23A (RPL 23A), ribosomal protein L17 (RPL 17), ribosomal protein L24 (RPL 24), ribosomal protein L26 (RPL 26), ribosomal protein L27 (RPL 27), ribosomal protein L30 (RPL 30), ribosomal protein L27A (RPL 27A), ribosomal protein L28 (RPL 28), ribosomal protein L, ribosomal protein L29 (RPL 29), ribosomal protein L31 (RPL 31), ribosomal protein L32 (RPL 32), ribosomal protein L35A (RPL 35A), ribosomal protein L37 (RPL 37), ribosomal protein L37A (RPL 37A), ribosomal protein L38 (RPL 38), ribosomal protein L39 (RPL 39), ribosomal protein large P0 (RPLP 0), ribosomal protein large P1 (RPLP 1), ribosomal protein large P2 (RPLP 2), ribosomal protein S3 (RPS 3), ribosomal protein S3A (RPS 3A), ribosomal protein, X-linked ribosomal protein S4 (RPS 4X), Y-linked ribosomal protein S41 (RPS 4Y 1), ribosomal protein S5 (RPS 5), ribosomal protein S6 (RPS 6), ribosomal protein S7 (RPS 7), ribosomal protein S8 (RPS 8), ribosomal protein S9 (RPS 9), ribosomal protein S10 (RPS 10), ribosomal protein S11 (RPS 11), ribosomal protein S12 (RPS 12), ribosomal protein S13 (RPS 13), ribosomal protein S15 (RPS 15), ribosomal protein S15A (RPS 15A), ribosomal protein S10 (RPS 10), Ribosomal protein S16 (RPS 16), ribosomal protein S19 (RPS 19), ribosomal protein S20 (RPS 20), ribosomal protein S21 (RPS 21), ribosomal protein S23 (RPS 23), ribosomal protein S25 (RPS 25), ribosomal protein S26 (RPS 26), ribosomal protein S27 (RPS 27), ribosomal protein S27a (RPS 27 a), ribosomal protein S28 (RPS 28), ribosomal protein S29 (RPS 29), ribosomal protein L15 (RPL 15), ribosomal protein S2 (RPS 2), ribosomal protein S27a (RPS 27a, Ribosomal protein L14 (RPL 14), ribosomal protein S14 (RPS 14), ribosomal protein L10 (RPL 10), ribosomal protein L10A (RPL 10A), ribosomal protein L35 (RPL 35), ribosomal protein L13A (RPL 13A), ribosomal protein L36 (RPL 36), ribosomal protein L36A (RPL 36A), ribosomal protein L41 (RPL 41), ribosomal protein S18 (RPS 18), ribosomal protein S24 (RPS 24), ribosomal protein L8 (RPL 8), ribosomal protein L34 (RPL 34), and, Ribosomal protein S17 (RPS 17), ribosomal Protein SA (RPSA), or ribosomal protein S17 (RPS 17), or a sequence of a gene encoding a ribosomal protein having at least, up to, just below, or between any two of the foregoing (inclusive or exclusive) identity to any of the ribosomal gene protein sequences 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. in some embodiments, 1, 2, 3,4, 5 or more of the foregoing 3' utr sequences may be excluded from the RNA molecules disclosed herein.

In some embodiments, the 3'UTR comprises a sequence from the 3' UTR region of a gene encoding a ribosomal protein or from a gene comprising ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA 52), widely expressed (FAU) Finkerr-Bischoff-lisril (Finkel-Biskis-Reilley) murine sarcoma virus (FBR-MuSV), ribosomal protein L22-like 1 (RPL 22L 1), ribosomal protein L39-like (RPL 39L), ribosomal protein L10-like (RPL 10L), ribosomal protein L36 a-like (RPL 36 AL), ribosomal protein L3-like (RPL 3L), ribosomal protein S27-like (RPS 27L), ribosomal protein L26-like 1 (RPL 26L 1), ribosomal protein L7-like 1 (RPL 7L 1), ribosomal protein L13a pseudogene (RPL 13 AP), ribosomal protein L37a pseudogene 8 (RPL 37AP 8), ribosomal protein S10-like (RPL 36A), ribosomal protein L26P 5, ribosomal protein L26P 14 (RPP 6), ribosomal protein P5P 14 (RPP 6, and the like (RPP 6P 36P 14); and/or a sequence of 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% of a gene encoding a protein having at least, up to, just below, or between any two of the foregoing gene protein sequences (inclusive or exclusive). In some embodiments, 1,2, 3,4, 5 or more of the foregoing 3' utr sequences may be excluded from the RNA molecules disclosed herein.

It will be appreciated by those of ordinary skill in the art that heterologous and/or synthetic 5 'UTRs can be used with any desired 3' UTR sequences, and vice versa. For example, heterologous 5 'UTRs can be used with synthetic and/or heterologous 3' UTRs.

In one embodiment, the 3' UTR encoding DNA disclosed herein comprises a sequence 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to, at least, up to, just below, or between any two of the following (inclusive or exclusive) SEQ ID NO. 20. In one embodiment, the DNA encoding the 3' UTR comprises the sequence of SEQ ID NO: 20. In some embodiments, the RNAs disclosed herein comprise 3 'utrs comprising sequences having at least, up to, just below, or between any two of (inclusive or exclusive) identity to the 3' utrs provided in either of SEQ ID NOs 21 or 22, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one embodiment, the 3' UTR comprises the sequence of either of SEQ ID NOs 21 or 22.

【SEQ ID NO:20(DNA)】

【SEQ ID NO:21(RNA)】

【SEQ ID NO:22(RNA)】

In one embodiment, the 3' UTR encoding DNA disclosed herein comprises a sequence 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to, at least, up to, just below, or between any two of (inclusive or exclusive of) SEQ ID NO. 23. In one embodiment, the DNA encoding the 3' UTR comprises the sequence of SEQ ID NO. 23. In one embodiment, the RNAs disclosed herein comprise 3 'utrs comprising sequences having at least, up to, just below, or between any two of the following, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the 3' utrs provided in any one of SEQ ID NOs 24 or 25. In one embodiment, the 3' UTR comprises the sequence of either one of SEQ ID NOs 24 or 25.

【SEQ ID NO:23(DNA)】

【SEQ ID NO:24(RNA)】

【SEQ ID NO:25(RNA)】

In some embodiments, 1, 2, 3,4, 5 or more of the foregoing 3' utr sequences may be excluded from the RNA molecules disclosed herein.

[ D ] Open Reading Frame (ORF) ]

The 5 'and 3' UTRs are operably linked to an Open Reading Frame (ORF), which may be a codon sequence capable of translation into a polypeptide of interest. The open reading frame may be the sequence of several DNA or RNA nucleotide triplets, which can be translated into a peptide or protein. The ORF may begin with an initiation codon at its 5' end and at the next region, for example a combination of three consecutive nucleotides (ATG or AUG) typically encoding the amino acid methionine, which is typically a multiple of 3 nucleotides in length. The open reading frame may end with at least one stop codon including, but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some embodiments, the open reading frame may end with one, two, three, four, or more stop codons, including but not limited to TAATAA(SEQ ID NO：27)、TAATAG(SEQ ID NO：28)、TAATGA(SEQ ID NO：29)、TAGTGA(SEQ ID NO：30)、TAGTAA(SEQ ID NO：31)、TAGTAG(SEQ ID NO：32)、TGATGA(SEQ ID NO：33)、TGATAG(SEQ ID NO：34)、TGATAA(SEQ ID NO：35) or UAAUAA(SEQ ID NO：36)、UAAUAG(SEQ ID NO：37)、UAAUGA(SEQ ID NO：38)、UAGUGA(SEQ ID NO：39)、UAGUAA(SEQ ID NO:40)、UAGUAG(SEQ ID NO：41)、UGAUGA(SEQ ID NO：42)、UGAUAG(SEQ ID NO：43)、UGAUAA(SEQ ID NO：44), or any combination thereof. The open reading frame may be isolated, or it may be incorporated into a longer nucleic acid sequence, for example into a vector or mRNA. The open reading frame may also be referred to as a "(protein) coding region" or "coding sequence".

As set forth herein, an RNA molecule may include one (monocistronic), two (bicistronic), or more (polycistronic) open reading frames.

In some embodiments, the ORF encodes a non-structural viral gene. In some embodiments, the ORF further comprises one or more subgenomic promoters. In some embodiments, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some embodiments, the first RNA molecule does not include an ORF encoding any polypeptide of interest, and the second RNA molecule includes an ORF encoding a polypeptide of interest. In some embodiments, the first RNA molecule does not include a subgenomic promoter.

The present invention provides an RNA molecule comprising at least one open reading frame encoding a Respiratory Syncytial Virus (RSV) polypeptide. In some embodiments, the RNA molecule comprises at least one open reading frame encoding an RSV F protein. In a preferred embodiment, the RNA molecule comprises at least one open reading frame encoding a Respiratory Syncytial Virus (RSV) pre-fusion F protein (preF) polypeptide.

[ E. Gene of interest ]

The RNA molecules described herein can include a gene of interest. The gene of interest encodes a polypeptide of interest. Non-limiting examples of polypeptides of interest include, for example, biological agents, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane binding polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, those polypeptides encoded by human genomes for which no therapeutic indications have been determined but which are useful in research and exploration areas, or combinations thereof. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing polypeptides of interest may be excluded. The sequences of specific genes of interest are readily available to those skilled in the art using public and private databases (e.g) And (5) identification.

In some embodiments, the RNA molecule comprises a coding region for a gene of interest. In some embodiments, the gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or immunogenic fragment thereof. In some embodiments, the antigenic polypeptide comprises one epitope from an antigen. In some embodiments, the antigenic polypeptide comprises a plurality of different epitopes from an antigen. In some embodiments, an antigenic polypeptide comprising a plurality of different epitopes from an antigen is multi-epitope. In some embodiments, the antigenic polypeptide comprises an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide. In some embodiments, 1, 2, 3, 4,5, or more of the foregoing antigenic polypeptides may be excluded.

The term "antigen" may refer to a substance that is capable of being recognized by the immune system (e.g., by the acquired immune system) and of eliciting an antigen-specific immune response, e.g., by forming antibodies and/or antigen-specific T cells, as part of the acquired immune response. The antigen may be or may comprise a peptide or protein that may be presented by MHC to T cells. An antigen may be a translation product of a provided nucleic acid molecule (e.g., an RNA molecule comprising at least one coding sequence as described herein). In addition, fragments, variants and derivatives of an antigen (such as a peptide or protein) comprising at least one epitope are understood as antigens.

In some embodiments, the RNA encoding the gene of interest (e.g., antigen) is expressed in cells of the treated individual to provide the gene of interest (e.g., antigen). In some embodiments, the RNA is transiently expressed in cells of the individual. In some embodiments, the expression of the gene of interest (e.g., antigen) is at the cell surface. In some embodiments, the gene of interest (e.g., antigen) is expressed and presented in the context of MHC. In some embodiments, the gene of interest (e.g., antigen) is expressed into the extracellular space, e.g., the antigen is secreted.

In some embodiments, the RNA molecule includes a coding region for a gene of interest (e.g., an antigen). In some embodiments, the RNA molecule includes a coding region for a gene of interest (e.g., an antigen) derived from a pathogen associated with an infectious disease. In some embodiments, the RNA molecule includes a coding region derived from a gene of interest (e.g., antigen) of RSV.

In some embodiments, the RNA molecule encodes RSV preF protein, or a fragment or variant thereof.

In some embodiments, an RNA polynucleotide described herein, or a composition or pharmaceutical formulation comprising the same, comprises a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some embodiments, an RNA polynucleotide described herein or a composition or pharmaceutical formulation comprising the same is transcribed from a DNA template. In some embodiments, the DNA template for transcription of an RNA polynucleotide described herein comprises a sequence complementary to the RNA polynucleotide. In some embodiments, a gene of interest described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide encodes a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some embodiments, the polypeptides described herein are encoded by RNA polynucleotides transcribed from a DNA template comprising a sequence complementary to the RNA polynucleotides.

In some embodiments, the RNA molecule encodes RSV preF protein, and the RSV preF protein comprises the sequence of any one of SEQ ID NOs 1-6 and 71-74, or a fragment or variant thereof.

In some embodiments, the RNA molecule encodes a RSV preF protein synthesized from a nucleic acid sequence comprising any one of SEQ ID NOs 7-10 and 59-62, or a fragment or variant thereof.

[ F. multiple A tails ]

In some embodiments, the RNA molecules disclosed herein comprise a polyadenylation (poly a) sequence, e.g., as described herein. In some embodiments, the poly-a sequence is located downstream of the 3'utr, e.g., adjacent to the 3' utr. "poly A tail" or "poly A sequence" refers to a contiguous stretch of adenine residues that may be attached to the 3' end of an RNA molecule, for example, having up to or about 400 adenosine nucleotides in length, for example, or about 20 to about 400, preferably or about 50 to about 400, more preferably or about 50 to about 300, even more preferably or about 50 to about 250, and most preferably or about 60 to about 250 adenosine nucleotides. Poly a sequences are known to those skilled in the art and can be appended to the 3' utr in the RNA molecules described herein. Multiple a tails may increase the stability, half-life, and/or translational efficiency of RNA molecules.

After cleavage, most of the pre-mRNA obtained a polyadenylation tail, with the exception of pre-mRNA from replication-dependent histone transcripts ending with histone stem-loop substitutions for multiple A sequences. In this context, the 3' end is processed as a nuclear co-transcription process that facilitates the transport of mRNA from the nucleus to the cytoplasm and affects mRNA stability and translation. The formation of this 3' end occurs in a two-step reaction directed by a cleavage/polyadenylation mechanism and depends on the two sequence components in the pre-mRNA, the hexanucleotide polyadenylation signal and the presence of downstream G/U-rich sequences. In a first step, the pre-mRNA between these two modules is cleaved into the free 3' hydroxyl groups. In the second step, the newly formed 3' end is extended by polyadenylation or addition of a poly A sequence.

Polyadenylation refers to the addition of multiple a sequences to an RNA molecule, for example to immature mRNA. Polyadenylation may be induced by so-called polyadenylation signals. This signal may be located close to the 3 'end of the RNA molecule to be polyadenylation or within the nucleotide extension at its 3' end. The 3' UTR of the artificial nucleic acid molecule may also comprise polyadenylation signals. The polyadenylation signal generally comprises a hexamer, preferably the hexamer sequence AAUAAA, consisting of adenine and uracil/thymine nucleotides, although other sequences (preferably hexamer sequences) are also contemplated. Polyadenylation generally occurs during the processing of pre-mRNA (also known as immature mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) involves a polyadenylation step. Multiple a tailing of mRNA transcribed in vitro can be accomplished using a variety of approaches including, but not limited to, cloning of poly-T segments into DNA templates, or post-transcriptional addition by use of a multiple a polymerase. The term may refer to polyadenylation of RNA as a cellular process, or to polyadenylation by in vitro enzymatic reactions with suitable enzymes such as E.coli (E.coli) poly-A polymerase, or by chemical synthesis.

The RNA molecules disclosed herein can have a poly-a sequence linked to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or a poly-a sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. In some embodiments, the multiple a sequences are linked during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidines) in the strand complementary to the coding strand.

The DNA sequence (coding strand) encoding the multiple A sequence is called the multiple A cassette. In some embodiments, the multiple a cassettes present in the coding strand of the DNA consist essentially of dA nucleotides, but are interspersed with random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences may be at least, up to, just below, or between any two of the following (inclusive or exclusive) nucleotides ：5、6、7、8、9、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 or 50 in length. Such a cartridge is disclosed, for example, in WO 2016/005324A1 (which is incorporated herein by reference). Any of the multiple A cassettes disclosed in WO 2016/005324A1 may be used in the present invention. Multiple a cassettes consisting essentially of dA nucleotides but interspersed with random sequences of four nucleotides (dA, dC, dG, dT) equally distributed and for example 5 to 50 nucleotides in length show continued propagation of plasmid DNA in e.coli at the DNA level and still correlate at the RNA level with beneficial properties with respect to supporting RNA stability and translation efficiency. In some embodiments, the poly-a sequence contained in the RNA polynucleotides described herein consists essentially of adenosine nucleotides, but is interspersed with random sequences of four nucleotides (A, C, G, U). Such random sequences may be at least, up to, just below, or between any two of the following (inclusive or exclusive) nucleotides ：5、6、7、8、9、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 or 50 in length.

The poly-A sequence may be located at any position within the 3' UTR. In some embodiments, no nucleotide other than an adenosine nucleotide flanks the poly-a sequence at the 3' end of the poly-a sequence, e.g., the 3' end of the poly-a sequence is not masked by a nucleotide other than adenosine or the 3' end of the poly-a sequence is not followed by a nucleotide other than adenosine. In some embodiments, the poly-a sequence may be located at the 3 'end of the 3' utr, e.g., the 3'utr does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 nucleotide located 3' of the poly-a sequence, more preferably the 3'utr does not contain other components located 3' of the poly-a sequence. In some embodiments, the poly-a sequence is located at the 3 'end of the RNA molecule, e.g., the artificial nucleic acid molecule does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 nucleotide located 3' of the poly-a sequence. Alternatively, the poly-A sequence may be located at the 5' end of the 3' UTR, e.g., located immediately 3' of the ORF of the artificial nucleic acid molecule, or within the 3' UTR, e.g., flanking the 5' and 3' sides of other 3' UTR components. In some embodiments, the poly-a sequence is flanked 3' to the poly-C sequence and/or the histone stem-loop sequence. Alternatively or additionally, the poly-a sequence may flank the 5 'side of a 3' utr module derived from, for example, a human albumin or globulin gene.

In some embodiments, the RNA molecule can further include an endonuclease recognition site sequence immediately downstream of the poly-a tail sequence. The RNA molecule can further include a poly a polymerase recognition sequence (e.g., polyadenylation signal) (e.g., AAUAAA) near its 3' end. In some embodiments, the polyadenylation signal is located 3 'of the poly a sequence contained in the 3' utr. In some embodiments, the poly-a sequence is separated from the polyadenylation signal by ：1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145 or 150, wherein the nucleotide sequence preferably does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides, by a nucleotide sequence comprising or consisting of at least, up to, just below, or between any two of the following (inclusive or exclusive). In some embodiments, the nucleotide sequence separating the poly-a sequence from the polyadenylation signal comprises or comprises about 1 to about 200 nucleotides, such as 10 to 90, 20 to 85, 30 to 80, 40 to 80, 50 to 75, or 55 to 85 nucleotides, more preferably 55 to 80 nucleotides, and the nucleotide sequence does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.

In some embodiments, the polyadenylation signal comprises the consensus sequence NN (U/T) ANA, where n=a or U, preferably AA (U/T) AAA or a (U/T) AAA. Such consensus sequences can be recognized by most animal and bacterial cell systems, for example by polyadenylation factors such as cleavage/polyadenylation specific factors (CPSF) in conjunction with CstF, PAP, PAB, CFI and/or CFII. In some embodiments, the polyadenylation signal (e.g., consensus NNUANA) is located less than or less than about 50 nucleotides downstream of the 3' end of the 3' utr module defined herein, e.g., at least, up to, just below, or between any two of (including or exclusive of) 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides, such that transcription of the RNA molecule will produce an immature RNA containing the polyadenylation signal downstream of its 3' utr and subsequently ligating the poly a sequence to the immature RNA. Thus, the resulting RNA can comprise a 3'utr comprising at least one poly a sequence, and wherein the 3' utr is followed by an additional poly a sequence.

The poly a sequence may have any length. In some embodiments, the length of the poly-A tail can be 5 to 300 nucleotides. In some embodiments, the RNA molecule comprises, consists essentially of, or consists of a sequence of or about 25 to about 400 adenosine nucleotides, a sequence of or about 50 to about 300 adenosine nucleotides, a sequence of or about 50 to about 250 adenosine nucleotides, a sequence of or about 60 to about 250 adenosine nucleotides, or a sequence of or about 40 to about 100 adenosine nucleotides. In some embodiments, the poly a tail comprises, consists essentially of, or consists of ：5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、500、505、510、515、520、525、530、535、540、545、550、555、560、565、570、575、580、585、590、595、600、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895、900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、980、985、990、995 or 1000 adenosine nucleotides at least, up to, just below, or between any two of the following (inclusive or exclusive). In this context, "consisting essentially of" means that a majority of the nucleotides in the poly-a sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the number of nucleotides in the poly-a sequence are adenosine nucleotides, but allows the remaining nucleotides to be nucleotides other than adenosine nucleotides, such as uridine, guanosine and/or cytosine. In this context, "consisting of" means that all nucleotides in the poly-a sequence (i.e., 100% of the number of nucleotides in the poly-a sequence) are adenosine nucleotides.

In some embodiments, the RNA molecule includes a multi-a tail comprising a sequence of greater than 30 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly a tail comprising or containing about 40 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly a tail comprising or containing about 80 adenosine nucleotides. In some embodiments, the 3' poly a tail has an extension of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some specific embodiments, the RNA molecule comprises or comprises about 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises or includes about 80 consecutive adenosine residues. Multiple a tails can play a key regulatory role in enhancing translation efficiency and regulating mRNA quality control and degradation efficiency. Short sequence or superadenosine can degrade signaling RNAs.

In some embodiments, the multi-a tail can be located within an RNA molecule or other nucleic acid molecule, such as in a vector, e.g., in a vector that serves as a template for the production of RNA (e.g., mRNA) from, for example, a transcription vector. In some embodiments, the RNA molecule may not include a multi-a tail.

In one embodiment, the DNA encoding the multiple A tails disclosed herein comprises a sequence 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to SEQ ID NO 26, at least, up to, just below, or between any two of the following (inclusive or exclusive). In one embodiment, the DNA encoding the multiple A tails comprises the sequence of SEQ ID NO. 26. In one embodiment, the RNAs disclosed herein comprise a multi-Atail comprising a sequence that has at least, up to, just below, or between any two of (inclusive or exclusive) identity to SEQ ID NO:26, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one embodiment, the multiple A tail comprises the sequence of SEQ ID NO. 26. In one embodiment, the poly A tail comprises a sequence of 26.+ -.2 adenosine (A) nucleotides of SEQ ID NO. In one embodiment, the poly A tail comprises a sequence of 26.+ -.1 adenosine (A) nucleotides of SEQ ID NO. In one embodiment, the multiple A tail comprises the sequence of SEQ ID NO. 26. In one embodiment, the poly A tail comprises a sequence of 26.+ -.2 adenosine (A) nucleotides of SEQ ID NO. In one embodiment, the poly A tail comprises a sequence of 26.+ -.1 adenosine (A) nucleotides of SEQ ID NO. In some embodiments, the poly A tail comprises the sequence of SEQ ID NO. 26.

In some embodiments, 1,2, 3,4, 5 or more of the foregoing poly-a sequences may be excluded from the RNA molecules disclosed herein.

【SEQ ID NO:26(DNA,RNA)】

AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

[ G. other Components ]

In some embodiments of the invention, the RNA molecule further comprises a chain terminating nucleoside. For example, chain terminating nucleosides can include those that are deoxy at the 2 'and/or 3' positions of their glycosyl groups. Such species may include 3' -deoxyadenosine (cordycepin (cordycepin)), 3' -deoxyuridine, 3' -deoxycytosine, 3' -deoxyguanosine, 3' -deoxythymine, and 2',3' -dideoxynucleosides such as 2',3' -dideoxyadenosine, 2',3' -dideoxyuridine, 2',3' -dideoxycytosine, 2',3' -dideoxyguanosine, and 2',3' -dideoxythymine. In some embodiments, 1,2, 3,4,5 or more of the foregoing chain terminating nucleosides can be excluded from the RNA molecules disclosed herein. In some embodiments, the incorporation of a chain terminating nucleotide into the mRNA (e.g., at the 3' end) may result in stabilization of the mRNA, e.g., as described in international patent publication No. WO 2013/103659.

In some embodiments of the invention, the RNA molecule additionally comprises a stem loop, such as a histone stem loop. The stem loop may comprise 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, the stem loop may comprise 4, 5, 6, 7, or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in, before or after the untranslated region (5 'UTR or 3' UTR), the coding region, or the poly-A sequence or tail. In some embodiments, the stem loop may affect one or more functions of the mRNA, such as initiation of translation, translation efficiency, and/or transcription termination. Such histone stem-loop sequences may be those disclosed in WO 2012/019780 (the disclosure of which is incorporated herein by reference in its entirety). Other non-limiting examples of histone stem loop structures and nucleic acid sequences encoding such structures can be found, for example, in WO 2016/091391 (the disclosure of which is incorporated herein by reference in its entirety).

In some embodiments, the poly-a sequence or polyadenylation signal works synergistically with the combination of at least one histone stem-loop (even though both represent alternative mechanisms in nature) to increase the content observed in the case of protein expression beyond any of the individual components. In some embodiments, the synergy of the combination of multiple a and at least one histone stem-loop is independent of the order of the components and/or the length of the multiple a sequence.

In some embodiments, the RNA does not comprise a histone downstream component (histone downstream element; HDE). HDE comprises a naturally occurring stem-loop 3' approximately 15-20 nucleotide purine-rich polynucleotide extension, which represents the binding site of U7 snRNA, which involves processing histone pre-mRNA into mature histone mRNA.

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 inverted complementary sequences separated by a spacer, consisting of a short sequence, forming a structural loop. Unpaired loop regions are generally unable to base pair with any of the stem-loop components. The stability of the stem-loop structure is generally dependent on the length of the pairing region, the number of mismatches or bulges and/or 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 comprises 15 to 45 nucleotides in length.

In some embodiments, the RNA molecule comprises (e.g., within the 3' utr) a poly (C) sequence. In some embodiments, the poly-C sequence has at least, up to, just below, or between any two of (inclusive or exclusive) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 cytidines. In some embodiments, the poly-C sequence has or has about 30 cytidines.

In some embodiments, the RNA molecule includes an internal ribosome entry site (internal ribosome ENTRY SITE; IRES) sequence or IRES motif. In some embodiments, for example, if the RNA encodes two or more peptides or proteins, the IRES sequence separates the ORFs. IRES sequences may therefore be suitable if the RNA molecule is a bicistronic or polycistronic nucleic acid molecule.

In some embodiments, the RNA does not comprise an intron. In some embodiments, the RNA may alternatively or additionally comprise a microrna binding site.

Representative RNA molecules including combinations of the components disclosed herein may include, but are not limited to, the following in the 5 '-to-3' direction:

ORF-poly A sequence;

ORF-IRES-ORF-poly A sequence;

ORF-3' UTR-poly A sequence;

ORF-poly A sequence-3' UTR;

ORF-3' utr-poly a sequence-poly (C) sequence-histone stem-loop;

ORF-3' UTR-poly A sequence-poly (C) sequence-poly A sequence;

ORF-3' UTR-poly A sequence-histone stem-loop-poly A sequence;

5'UTR-ORF-3'UTR;

A 5' UTR-ORF-poly A sequence;

5' utr-ORF-poly a sequence-poly (C) sequence-histone stem-loop;

5' UTR-ORF-poly A sequence-poly (C) sequence-poly A sequence;

5' utr-ORF-poly a sequence-histone stem-loop-poly a sequence;

a 5'UTR-ORF-3' UTR-poly A sequence;

5'UTR-ORF-3' UTR-poly A sequence-poly (C) sequence

5'Utr-ORF-3' utr-poly a sequence-poly (C) sequence-histone stem-loop;

5' -cap-5 ' UTR-ORF-3' UTR;

a 5 '-cap-5' utr-ORF-poly a sequence;

A 5' -cap-5 ' UTR-ORF-3' UTR-poly A sequence;

5' -cap-5 ' UTR-ORF-3' UTR-poly A sequence-poly (C) sequence, or

5' -Cap-5 ' utr-ORF-3' utr-poly a sequence-poly (C) sequence-histone stem-loop.

In some embodiments, 1,2, 3, 4, 5 or more of the foregoing components may be excluded from the RNA molecules disclosed herein.

[ H. self-amplified RNA (saRNA) ]

In some embodiments, the RNA molecule may be saRNA. "self-amplifying RNA", "saRNA" and "replicon" refer to RNA capable of self-replication. Self-amplifying RNA molecules can be generated by using replication components derived from, for example, an alphavirus, and replacing the structural viral polypeptide with a nucleotide sequence encoding the polypeptide of interest. Self-amplifying RNA molecules are typically positive-strand molecules that can be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA causes the production of a plurality of daughter RNA molecules. These daughter RNA molecules, as well as collinear subgenomic transcripts, may themselves be translated to provide in situ expression of the encoded gene of interest (e.g., viral antigen), and/or may be transcribed to provide other transcripts synonymous with the delivered RNA translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is that the number of introduced saRNA molecules is amplified and thus the encoded gene of interest (e.g. viral antigen) becomes the main polypeptide product of the cell.

In some embodiments, the self-amplifying RNA includes at least one or more genes including any one of viral replicase, viral protease, viral helicase, and other non-structural viral proteins, or a combination thereof. In some embodiments, 1,2,3, or more of the foregoing genes may be excluded from the self-amplifying RNA molecules disclosed herein. In some embodiments, self-amplifying RNA may also include 5 'and 3' terminal pull-copy sequences, and optionally heterologous sequences encoding a desired amino acid sequence (e.g., an antigen of interest). Subgenomic promoters that direct expression of heterologous sequences may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., antigen of interest) may be fused in-frame to other coding regions in the self-amplifying RNA and/or may be under the control of an Internal Ribosome Entry Site (IRES).

In some embodiments, the self-amplifying RNA molecules described herein encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule, and (ii) a polypeptide of interest, such as a viral antigen. In some embodiments, the polymerase can be an alphavirus replicase, e.g., comprising any one of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, or any combination thereof. In some embodiments, 1,2,3, or more of the foregoing alphavirus proteins may be excluded from the RNA molecules disclosed herein.

In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5 ') open reading frame may encode a replicase and the second (3') open reading frame may encode a polypeptide comprising an antigen of interest. In some embodiments, the RNA can have an additional (e.g., downstream) open reading frame, e.g., to encode other antigens or to encode a helper polypeptide.

In some embodiments, the saRNA molecule further comprises (1) an alphavirus 5 'replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.

In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen, or optionally two or more polypeptide antigens, linked together (e.g., in tandem) in such a way that each of the sequences retains its identity when expressed as an amino acid sequence. The polypeptide produced from the self-amplified RNA can then be produced in the form of a fusion polypeptide or engineered in such a way as to produce an isolated polypeptide or peptide sequence.

In some embodiments, the self-amplifying RNAs described herein may encode one or more polypeptide antigens comprising a range of epitopes. In some embodiments, the self-amplifying RNA described herein may encode an epitope capable of eliciting a helper T cell response or a cytotoxic T cell response, or both.

In one embodiment, the self-amplifying RNA disclosed herein comprises a subgenomic promoter comprising a sequence having at least, up to, just below, or between any two of SEQ ID NO 54, 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one embodiment, the subgenomic promoter comprises the sequence of SEQ ID NO. 54.

【SEQ ID NO:54(RNA)】

CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAG

In some embodiments, the self-amplifying RNA molecules described herein encode (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule, and (ii) a polypeptide of interest, such as a viral antigen. In some embodiments, the polymerase can be an alphavirus replicase, e.g., including any one of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof.

In one embodiment, the self-amplifying RNA disclosed herein comprises an alphavirus replicase, e.g., comprising any one of the alphavirus proteins nsP1, nsP2, nsP3, nsP4 and any combination thereof, comprising a sequence having at least, up to, just below or any two of 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with SEQ ID NO 55-58, respectively. In one embodiment, the alphavirus proteins nsP1, nsP2, nsP3 and nsP4 each comprise the sequence of SEQ ID NOS 55-58, respectively.

【SEQ ID NO:55(nsP1 RNA)】

【SEQ ID NO:56(NSP2 RNA)】

【SEQ ID NO:57(NSP3 RNA)】

【SEQ ID NO:58(NSP4 RNA)】

[ IV. RNA transcription ]

In some embodiments, the RNAs disclosed herein are produced by in vitro transcription or chemical synthesis. In the context of the present invention, the term "transcription" relates to a process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into peptides or proteins.

According to the present invention, "transcription" includes "in vitro transcription" or "IVT" which refers to a process in which transcription occurs in vitro in a non-cellular system to produce synthetic RNA products for various applications, including, for example, the production of proteins or polypeptides. Methods for in vitro transcription of mRNA are well known in the art (see, e.g., losick, R.1972.In vitro transcription, ann Rev Biochem,41 409-46; kamakaka, R.T. and Kraus,W.L.2001.In vitro Transcription,Current Protocols in Cell Biology,2:11.6:11.6.1-11.6.17;Beckert,B. and Masquida,B.2010.Synthesis of RNA by In vitro Transcription in RNA,Methods in Molecular Biology,703(Neilson,H., et al, N.Y. Humana Press,2010; brunelle, J.L. and Green, R.2013, fifth chapter-In vitro transcription from plasmid or PCR-AMPLIFIED DNA, methods in Enzymology 530:101-114; all of which are incorporated herein by reference).

Cloning vectors can be used to generate transcripts. These cloning vectors are generally referred to as transcription vectors and are encompassed by the term "vector" according to the present invention. According to a particular embodiment, the RNA used is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. Template DNA can be prepared for in vitro transcription from a variety of sources using suitable techniques well known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see LINPINSEL, j.l and Conn, g.l., generalprotocols for preparation of PLASMID DNA TEMPLATE, and Bowman, j.c., azizi, b., lenz, t.k., ray, p.and Williams,L.D.,RNA in vitro transcription and RNA purification by denaturing PAGEin Recombinant andin vitro RNA syntheses,Methods 941Conn G.L.(), new York, n.y. Humana Press,2012, each of which is incorporated herein by reference). The promoter used to control transcription may be any promoter for any RNA polymerase. Specific examples of RNA polymerase are T7, T3 and SP6 RNA polymerase. Preferably, in vitro transcription according to the invention is under the control of the T7 or SP6 promoter. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

The synthetic IVT RNA product can be translated in vitro or introduced directly into a cell in which the product can be translated. In the case of RNA, the term "expression" or "translation" refers to the process by which mRNA strands direct the assembly of amino acid sequences in the ribosomes of cells to produce peptides or proteins. Such synthetic RNA products include, but are not limited to, for example, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribonucleases, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, micronuclear RNA molecules, micronucleolar RNA molecules, and the like. In some embodiments, 1, 2, 3, 4, 5, or more of the aforementioned synthetic RNA products may be excluded. The IVT reaction typically utilizes a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase as described and/or utilized herein.

In some embodiments, mRNA is made by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some embodiments, the RNAs disclosed herein are in vitro transcribed RNAs (IVT-RNAs), and may be obtained by in vitro transcription of an appropriate DNA template. The promoter used to control transcription may be any promoter for any RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In some embodiments, the starting material of the IVT can include a linearized DNA template, a nucleotide, an rnase inhibitor, a pyrophosphatase, and/or a polymerase (e.g., T7 RNA polymerase). Nucleotides may be manufactured internally, available from suppliers, or may be synthesized. Nucleotides can be, but are not limited to, those described herein, including natural and non-natural (modified) nucleotides. Any number of RNA polymerases or variants can be used, including but not limited to phage RNA polymerases, e.g., 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). In some embodiments, 1,2, 3,4, 5 or more of the foregoing RNA polymerases can be excluded. Some embodiments exclude the use of dnase.

In some embodiments, the IVT process is performed in a bioreactor. The bioreactor may comprise a mixer. In some embodiments, the nucleotides may be added to the bioreactor during the entire IVT process.

In some embodiments, in the bioreactor, one or more post-IVT agents are added to the IVT mixture comprising RNA after the IVT process. Exemplary post IVT agents may include dnase I configured to digest linearized DNA templates and/or proteinase K configured to digest dnase I and T7 RNA polymerase. In some embodiments, after the IVT, the post-IVT agent is incubated with the mixture in the bioreactor. In some embodiments, the bioreactor may contain at least, up to, just below, or between any two of the following (inclusive or exclusive) IVT mixture ：60、70、80、90、100、110、120、130、140、150,160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490 and 500 liters or more. The IVT mixture can have an RNA concentration ：3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、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、7.0、8.0、9.0、10、11、12、13、14、15、16、17、18、19、20、30、40、50、60、70、80、90 and 100mg/mL RNA or more that is or is not at least, up to, just below, or between any two of the following (inclusive or exclusive).

In some embodiments, the IVT mixture can include residual spermidine, residual DNA, residual protein, peptide, HEPES, EDTA, ammonium sulfate, cations (e.g., mg ²⁺、Na⁺、Ca²⁺), RNA fragments, residual nucleotides, free phosphate, or any combination thereof. In some embodiments, 1,2, 3, 4, 5, or more of the foregoing may be excluded from the IVT mixture.

Isolation and/or purification of nucleic acids described herein may include, but are not limited to, phenol/chloroform extraction and/or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid removal, quality assurance, and quality control. Additional non-limiting examples of purification procedures includeBeads (Beckman Coulter Genomics, danvers, mass.), poly-T beads, LNATM oligo-T Capture probesInc, vedbaek, denmark), 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), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits, including but not limited to the SV total separation system (Promega) and the in vitro transcription purge and concentrate kit (Norgen Biotek). In some embodiments, 1,2, 3, 4, 5, or more of the aforementioned purifications may be excluded.

When used in reference to a nucleic acid, the term "purified", such as "purified nucleic acid", refers to one that is separated from at least one contaminant. "contaminant (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 setting that is different from that which they find in nature, or in a form or setting that is different from that which exists prior to subjecting them to treatment and/or purification methods.

In some embodiments, at least a portion of the IVT mixture is filtered. The IVT mixture can be filtered via ultrafiltration and/or diafiltration to remove at least some impurities from the IVT mixture and/or to alter at least a portion of the buffer solution of the IVT mixture to produce a concentrated RNA solution as a retentate.

In some embodiments, "ultrafiltration" and "diafiltration" both refer to membrane filtration processes. Ultrafiltration generally uses membranes having a pore size of at least, up to, just below, or between any two of (inclusive or exclusive) 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm. In some embodiments, ultrafiltration membranes are generally classified by molecular weight cut-off (MWCO) rather than pore size. For example, the MWCO may be at least, up to, just below, or between any two of the following (including, or exclusive )：30kDa、40kDa、50kDa、60kDa、70kDa、80kDa、90kDa、100kDa、110kDa、120kDa、130kDa、140kDa、150kDa、160kDa、170kDa、180kDa、190kDa、200kDa、210kDa、220kDa、230kDa、240kDa、250kDa、260kDa、270kDa、280kDa、290kDa、300kDa、310kDa、320kDa、330kDa、340kDa、350kDa、360kDa、370kDa、380kDa、390kDa、400kDa、500kDa、600kDa、700kDa、800kDa、900kDa、1000kDa、2000kDa、3000kDa、4000kDa、5000kDa、6000kDa、7000kDa、8000kDa、9000kDa and 10000kDa. Those skilled in the art will appreciate that the filtration membrane may comprise different suitable materials including, for example, polymers, cellulose, ceramics, etc., depending on the application.

In some embodiments, ultrafiltration and diafiltration of the IVT mixture for purification of RNA may include (1) direct current filtration (DFF), also known as "dead-end" filtration, which applies a feed stream perpendicular to the membrane surface and attempts to pass 100% of the fluid through the membrane, and/or (2) Tangential Flow Filtration (TFF), also known as sweep filtration, in which the feed stream passes parallel to the membrane surface, with one portion passing through the membrane (permeate) and the remainder (retentate) retained and/or recycled back to the feed tank.

In some embodiments, filtration of the IVT mixture is performed via TFF comprising an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some embodiments, the first diafiltration step is performed in the presence of ammonium sulfate. The first diafiltration step may be configured to remove a majority of impurities from the IVT mixture. In some embodiments, the second diafiltration step is performed in the absence of ammonium sulfate. The second diafiltration step may be configured to transfer the RNA into the DS buffer formulation.

A filtration membrane with the appropriate MWCO may be selected for ultrafiltration in a TFF process. The MWCO of a TFF membrane determines which solutes can pass through the membrane into the filtrate and which solutes remain in the retentate. The MWCO of the TFF membrane may be selected such that substantially all solutes of interest (e.g., desired synthetic RNA species) remain in the retentate, rather than desired components (e.g., excess ribonucleotides, small nucleic acid fragments (such as digested or hydrolyzed DNA templates), peptide fragments (such as digested proteins), and/or other impurities) being transferred into the filtrate. In some embodiments, the retentate comprising the desired synthetic RNA species may be recycled into the feed tank for re-filtration in additional cycles. In some embodiments, the MWCO of the TFF membrane may be at least, up to, just below, or between (including or exclusive of) any two of 30kDa, 40kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, or more. In some embodiments, the MWCO of the TFF membrane may be at least, up to, just below, or between (including or exclusive of) any two of 100kDa, 150kDa, 200kDa, 250kDa, 300kDa, 350kDa, 400kDa, or more. In some embodiments, the MWCO of the TFF membrane may be at or about 250-350 kda. In some embodiments, the MWCO of the TFF membrane (e.g., cellulose-based membrane) may be or be about 30 to 300kDa, 50 to 300kDa, 100 to 300kDa, or 200 to 300kDa.

Diafiltration may be effected discontinuously or continuously. For example, in continuous diafiltration, diafiltration solution may be added to the sample feed reservoir at the same rate as the filtrate is produced. In this way, the volume of the sample reservoir remains constant, but small molecules (e.g., salts, solvents, etc.) that are free to permeate through the membrane are removed. Using solvent removal as an example, each additional percolation volume (DV) further reduces the solvent concentration. In discontinuous diafiltration, the solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of small molecules (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional Diafiltration Volume (DV) further reduces the concentration of small molecules (e.g., solvents). Continuous diafiltration generally requires a minimum volume for a given reduction of the molecules to be filtered. Discontinuous diafiltration, on the other hand, permits rapid changes in retentate conditions (such as pH, salt content, etc.). In some embodiments, the first diafiltration step is performed in an amount of at least, up to, just below, or between any two of (inclusive or exclusive) 2, 3, 4, 5, 6, 7, 8, 9, 10, or more diafiltration volumes. In some embodiments, the second diafiltration step is performed in an amount of at least, up to, just below, or between any two of (inclusive or exclusive) 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more diafiltration volumes. In some embodiments, the first diafiltration step is performed at 5 diafiltration volumes and the second diafiltration step is performed at 10 diafiltration volumes.

In some embodiments, for ultrafiltration and/or diafiltration, the IVT mixture is filtered ：100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、500、600、700、800、900 or 1000L/m ² filter area/hour or more at a rate of at least, up to, just below, or between any two of the following (inclusive or exclusive). The concentrated RNA solution can comprise at least, up to, just below, or between any two of (inclusive or exclusive) 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5mg/mL of single stranded RNA.

In some embodiments, the bioburden of the concentrated RNA solution from which the RNA product solution is obtained via filtration may also be reduced. Filtration may be performed using one or more filters for reducing bioburden. The one or more filters may include filters having a pore size of at least, up to, just below, or between any two of (inclusive or exclusive) 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.

As one example, reducing bioburden may include draining a retentate reservoir containing retentate obtained from ultrafiltration and/or diafiltration to obtain retentate. Reducing bioburden may include flushing the filtration system for ultrafiltration and/or diafiltration with a wash buffer solution to obtain a wash tank solution comprising residual RNA remaining in the filtration system. The retentate can be filtered to obtain a filtered retentate. The cell solution can be filtered using a first 0.2 μm filter to obtain a filtered cell solution. The retentate may be filtered using a first 0.2 μm filter or another 0.2 μm filter.

The filtered wash tank solution and the filtered retentate may be combined to form a combined tank solution. The combined pool solution can be filtered using a second 0.2 μm filter to obtain a filtered combined pool solution, which is further filtered using a third 0.2 μm filter to produce an RNA product solution.

Quality assurance and/or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, and/or analytical HPLC.

In some embodiments, the nucleic acid may be sequenced by methods including, but not limited to, reverse transcriptase-PCR.

In some embodiments, nucleic acids may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). Non-limiting examples of UV/Vis spectrometers areSpectrometer (thermo Fisher, waltham, mass.). Quantitative nucleic acids may be analyzed to determine if the nucleic acids may be of an appropriate size and/or to assess degradation. Degradation of nucleic acids can be assessed 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). In some embodiments, 1,2, 3, 4,5, or more of the foregoing assessment methods may be excluded.

V. RNA encapsulation

The RNA in the RNA product solution can be encapsulated, and the RNA solution can further comprise at least one encapsulating agent. In one embodiment, the encapsulating agent comprises a lipid, a Lipid Nanoparticle (LNP), a lipid complex, a polymer particle, a polymer complex, a monolithic delivery system, or a combination thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing components may be excluded as an encapsulant.

In one embodiment, the encapsulating agent is a lipid and results in Lipid Nanoparticle (LNP) -encapsulated RNA. Without intending to be bound by any theory, it is believed that the cationic or cationizable lipid or lipid-like material and/or cationic polymer combine with the nucleic acid to form aggregates, and that this aggregation produces colloidally stable particles.

The lipid may be a naturally occurring lipid or a synthetic lipid. However, lipids are typically biomass. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids and polymerizable lipids having ether and ester linked fatty acids, and combinations thereof. Lipids are substances which are insoluble in water and extractable with organic solvents. Compounds other than those specifically described herein are understood by those skilled in the art to be lipids and are encompassed by the compositions and methods of the present invention. The lipid components and non-lipids may be covalently or non-covalently linked to each other.

In some embodiments, the LNP can be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular rnases, and/or can be engineered for systemic delivery of RNA to target cells. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intravenously to an individual in need thereof. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intramuscularly to an individual in need thereof. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intradermally to an individual in need thereof. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intranasally to an individual in need thereof.

In one embodiment, the concentration of RNA in the RNA product solution is <1mg/mL. In another embodiment, the concentration of RNA is at least or at least about 0.05mg/mL. In another embodiment, the concentration of RNA is at least or at least about 0.5mg/mL. In another embodiment, the concentration of RNA is at least or at least about 1mg/ml. In another embodiment, the RNA concentration is at or about 0.05mg/mL to about 0.5mg/mL. In another embodiment, the concentration of RNA is at least 10mg/mL. In another embodiment, the concentration of RNA is at least 50mg/mL. In some embodiments, the concentration of RNA is or is not at least, up to, just below, between any two of the following (inclusive or exclusive), or about ：0.05mg/mL、0.5mg/mL、1mg/mL、10mg/mL、50mg/mL、75mg/mL、100mg/mL、150mg/mL、200mg/mL、250mg/mL、300mg/mL、400mg/mL or more below.

The present invention provides an RNA product solution and a lipid formulation mixture or composition thereof comprising at least one RNA encoding, for example, an antigen (e.g., pre-RSV fusion F protein), complexed with, encapsulated in and/or formulated with one or more lipids, and forming Lipid Nanoparticles (LNPs), liposomes, lipid complexes, and/or nanoliposomes. In some embodiments, the composition comprises lipid nanoparticles.

Lipid nanoparticle or LNP refers to any morphology of particles produced when cationic lipids and optionally one or more other lipids are combined, e.g., in an aqueous environment and/or in the presence of RNA. In some embodiments, the lipid nanoparticle is included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), to a target site of interest (e.g., cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticle of the invention comprises a nucleic acid (e.g., mRNA). Such lipid nanoparticles typically comprise a cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, polymer-bound lipids, or a combination thereof. In some embodiments, the LNP comprises at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., a steroid), and/or at least one polymer-bound lipid (e.g., a polyethylene glycol (PEG) modified lipid). In some embodiments, 1, 2,3, or more of the foregoing excipients may be excluded from the LNP.

In some embodiments, the LNP comprises 20-60mol% of one or more cationic (e.g., ionizable) lipids. For example, the LNP may comprise 20 to 50mol%, 20 to 40mol%, 20 to 30mol%, 30 to 60mol%, 30 to 50mol%, 30 to 40mol%, 40 to 60mol%, 40 to 50mol%, or 50 to 60mol% of one or more cationic (e.g., ionizable) lipids. In some embodiments, the LNP comprises or does not comprise at least, up to, just below, or between any two of the following (inclusive or exclusive) one or more cationic (e.g., ionizable) lipids, 20mol%, 30mol%, 40mol%, 50mol%, or 60mol%. In some embodiments, the LNP comprises 45 to 55 mole% (mol%) of one or more cationic (e.g., ionizable) lipids. For example, the LNP may or may not comprise at least, up to, just below, or between any two of the following (inclusive or exclusive) one or more cationic (e.g., ionizable) lipids 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mole%.

In some embodiments, the LNP comprises 5 to 25mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise 5 to 20mol%, 5 to 15mol%, 5 to 10mol%, 10 to 25mol%, 10 to 20mol%, 10 to 25mol%, 15 to 20mol%, or 20 to 25mol% of one or more neutral (e.g., non-cationic) lipids. In some embodiments, the LNP is or is not at least, up to, just below, or between any two of the following (inclusive or exclusive) one or more neutral (e.g., non-cationic) lipids: 5mol%, 10mol%, 15mol%, 20mol%, or 25mol%. In some embodiments, the LNP comprises 5 to 15mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise at least, up to, just below, or between any two of the following (inclusive or exclusive) of one or more neutral (e.g., non-cationic) lipids 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15mol%.

In some embodiments, the LNP comprises 25 to 55 mole% of one or more structural lipids (e.g., steroids). For example, the LNP may comprise 25～50mol％、25～45mol％、25～40mol％、25～35mol％、25～30mol％、30～55mol％、30～50mol％、30～45mol％、30～40mol％、30～35mol％、35～55mol％、35～50mol％、35～45mol％、35～40mol％、40～55mol％、40～50mol％、40～45mol％、45～55mol％、45～50mol％ or 50-55 mole% of one or more structural lipids (e.g., steroids). In some embodiments, the LNP is or is not at least, up to, just below, or between any two of (inclusive or exclusive) 25mol%, 30mol%, 35mol%, 40mol%, 45mol%, 50mol%, or 55mol% of one or more structural lipids (e.g., steroids). In some embodiments, the LNP comprises 35 to 40mol% of one or more structural lipids (e.g., steroids). For example, the LNP may comprise at least, up to, just below, or between any two of the following (inclusive or exclusive) one or more structural lipids (e.g., steroids): 35, 36, 37, 38, 39, or 40mol%.

In some embodiments, the LNP comprises 0.5 to 15mol% of one or more polymer-bound lipids (e.g., polyethylene glycol (PEG) modified lipids). For example, the lipid nanoparticle may comprise 0.5 to 10mol%, 0.5 to 5mol%, 1 to 15mol%, 1 to 10mol%, 1 to 5mol%, 2 to 15mol%, 2 to 10mol%, 2 to 5mol%, 5 to 15mol%, 5 to 10mol%, or 10 to 15mol% of one or more polymer-bound lipids (e.g., polyethylene glycol (PEG) -modified lipids). In some embodiments, the lipid LNP is or is not at least, up to, just below, or between any two of (inclusive or exclusive) 0.5mol%, 1mol%, 2mol%, 3mol%, 4mol%, 5mol%, 6mol%, 7mol%, 8mol%, 9mol%, 10mol%, 11mol%, 12mol%, 13mol%, 14mol%, or 15mol% of one or more polymer-bound lipids (e.g., polyethylene glycol (PEG) modified lipids). In some embodiments, the LNP comprises 1-2 mol% of one or more polymer-bound lipids (e.g., polyethylene glycol (PEG) modified lipids). For example, the LNP can comprise at least, up to, just below, or between any two of the following (inclusive or exclusive) 1, 1.5, or 2mol% of one or more polymer-bound lipids (e.g., polyethylene glycol (PEG) modified lipids).

In some embodiments, the LNP comprises: 20 to 75mol% of one or more cationic (e.g., ionizable) lipids (e.g., at least, up to, just below, or between any two of the following (inclusive or exclusive): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% and 75%), 0.5 to 25% by mole (e.g., at least, up to, just below, or between (including or exclusive of) one or more neutral (e.g., non-cationic) lipids, 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25% and 25%), 5 to 55% by mole (e.g., at least, up to, just below, or between (including or exclusive of) 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% and 55%) and 0.5 to 20% by mole (e.g., at least, up to, just below, or between (including or exclusive of) 0.5%, 2.5%, 3.5%, 6.5%, 9.5%, 5%, 17%, 11.5%, 15% by mole (including or exclusive of) one or more structured lipids (e.g., lipids such as polyethylene glycol (PEG) modified lipids). In some embodiments, 1,2, 3, or more of the lipids may be excluded from the LNP.

In some non-limiting embodiments, the molar lipid ratio was 50/10/38.5/1.5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 60/7.5/31/1.5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 57.5/7.5/31.5/3.5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 57.2/7.1/34.3/1.4 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid) 40/15/40/5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 50/10/35/4.5/0.5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 50/10/35/5 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 40/10/40/10 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), 35/15/40/10 (mol% cationic lipid/neutral lipid/structured lipid/polymer bound lipid), or 52/13/30/5 (mol% cationic lipid/neutral lipid/structural lipid/polymer-bound lipid).

In some embodiments, an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle and/or in an aqueous space encapsulated by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism or cell's mechanisms, such as an adverse immune reaction. Nucleic acids (e.g., mRNA) or portions thereof may also be bound to and complexed with lipid nanoparticles. The lipid nanoparticle may comprise any lipid capable of forming a particle that is linked to a nucleic acid and/or encapsulates one or more nucleic acids.

In some embodiments, provided RNA molecules (e.g., saRNA, mRNA) can be formulated with LNP. In some embodiments, the lipid nanoparticles may or may not have an average diameter of about 1-500 nm (e.g., at least, up to, just below, or between (including or exclusive )：1、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、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490 or 500 nm) in some embodiments, the average diameter of the lipid nanoparticles is or is about 30nm to about 150nm, about 40nm to about 150nm, about 50nm to about 150nm, about 60nm to about 130nm, about 70nm to about 110nm, about 70nm to about 100nm, about 80nm to about 100nm, about 90nm to about 100nm, about 70 to about 90nm, about 80nm to about 90nm, about 70nm to about 80nm, or at least, up to, just below, or between (including or exclusive )：30nm、35nm、40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150nm, and substantially non-toxic, the term "average diameter" is the average diameter of the fluid dimensions measured by Dynamic Light Scattering (DLS) and data analysis using a so-called cumulant algorithm (DLS) and the average diameter of the fluid dimensions measured by the data (average diameter, p.d. the average diameter, p.d. 2, p.m. of particles are provided by the average diameter, p.d. 2, p.m. of particles, p.p.m. 2, p.p.p.m. of the average diameter, p.p.p.2, p.f.f. of particles, p.w.10.p.m.

The LNPs described herein can exhibit a polydispersity index of less than or less than about 0.5, 0.4, 0.3, or 0.2 or less. By way of example, the LNP may or may not exhibit a polydispersity index ：0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49 or 0.5 of at least, up to, just below, or between (inclusive or exclusive) the following. In some embodiments, the polydispersity index is calculated based on dynamic light scattering measurements from a so-called cumulative analysis referred to in the definition of "average diameter". Under certain preconditions, it may be considered as a measure of the size distribution of the nanoparticle population.

In some embodiments, the LNP of the invention includes or does not include an N to P ratio of about 2:1 to about 30:1, such as an N to P ratio ：2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1 or 30:1 of at least, up to, just below, or between (inclusive or exclusive) of the following. In some embodiments, the LNP of the invention comprises an N to P ratio of at or about 6:1. In some embodiments, the LNP of the invention comprises an N to P ratio of at or about 3:1.

In some embodiments, the LNP of the invention comprises or does not comprise a wt/wt ratio of cationic lipid component to RNA of about 5:1 to about 100:1, such as at least, up to, just below, or between (inclusive or exclusive) wt/wt ratio ：5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1、54:1、55:1、56:1、57:1、58:1、59:1、60:1、61:1、62:1、63:1、64:1、65:1、66:1、67:1、68:1、69:1、70:1、71:1、72:1、73:1、74:1、75:1、76:1、77:1、78:1、79:1、80:1、81:1、82:1、83:1、84:1、85:1、86:1、87:1、88:1、89:1、90:1、91:1、92:1、93:1、94:1、95:1、96:1、97:1、98:1、99:1 or 100:1 of cationic lipid component to RNA. In some embodiments, the LNP of the invention comprises a wt/wt ratio of ionizable cationic lipid component to RNA of at or about 20:1. In some embodiments, the LNP of the invention comprises a wt/wt ratio of ionizable cationic lipid component to RNA of at or about 10:1.

In certain embodiments, the nucleic acid (e.g., RNA molecule) (when present in the provided LNP) is resistant to degradation with nucleases in aqueous solution. In some embodiments, the LNP is a liver-targeted lipid nanoparticle. In some embodiments, the LNP is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic LNP can comprise at least one cationic lipid, at least one polymer-bound lipid, and at least one helper lipid (e.g., at least one neutral lipid).

In certain embodiments, the RNA solution and lipid formulation mixtures or compositions thereof may have at least, up to, just below, between (including or exclusive of) or about below a particular lipid, lipid type, or non-lipid component (such as a lipid-like material and/or a cationic polymer and/or an adjuvant), antigen, peptide, polypeptide, sugar, nucleic acid, or other material ：1％、2％、3％、4％5％、6％、7％、8％、9％、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％、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% disclosed herein or that would be known to one of skill in the art.

The LNPs described herein can be produced using components, compositions, and methods generally known in the art, see, e.g., PCT/US2016/052352、PCT/US2016/068300、PCT/US2017/037551、PCT/US2015/027400、PCT/US2016/047406、PCT/US2016000129、PCT/US2016/014280、PCT/US2016/014280、PCT/US2017/038426、PCT/US2014/027077、PCT/US2014/055394、PCT/US2016/52117、PCT/US2012/069610、PCT/US2017/027492、PCT/US2016/059575 and PCT/US2016/069491, all of which are incorporated herein by reference in their entirety. Other non-limiting examples of methods for preparing LNP can be found, for example, in WO 2022/034154, the disclosure of which is incorporated herein by reference in its entirety.

For example, a method of preparing LNP can involve obtaining a colloid from at least one cationic or cationizable lipid or lipid-like material and/or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain a nucleic acid particle. As used herein, the term "colloid" refers to a homogeneous mixture in which the dispersed particles do not precipitate out. The insoluble particles in the mixture are tiny, with a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture and not the entire suspension.

In order to prepare a colloid comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer, conventional methods for preparing liposome vesicles may be applied herein and adapted appropriately. The most common methods for preparing liposome vesicles share the basic stages of (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using a variety of aqueous media).

In the membrane hydration method, lipids are first dissolved in a suitable organic solvent and dried to give a thin film at the bottom of the flask. The lipid membrane obtained is hydrated using a suitable aqueous medium, yielding a liposome dispersion. In addition, additional downsizing steps may be included.

Reverse phase evaporation is an alternative method of membrane hydration for the preparation of liposome vesicles, which involves the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Short sonication of this mixture is required for system homogenization. The organic phase was removed under reduced pressure to give a milky white gel, which was subsequently converted into a liposome suspension.

The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This procedure disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, such as liposome formation. In general, the RNA lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using ethanol injection techniques, in some embodiments, such colloidal liposome dispersions are formed by injecting an ethanol solution comprising lipids (such as cationic lipids and additional lipids) into an aqueous solution with stirring. In some embodiments, the RNA lipid complex particles described herein can be obtained without an extrusion step. The term "extrusion (extruding)" or "extrusion" refers to the production of particles having a fixed cross-sectional profile. In particular, it refers to reducing the size of the particles, wherein the particles are forced through a filter having defined pores.

Other methods for preparing colloids characterized by the absence of organic solvents may also be used in accordance with the present invention.

In some embodiments, LNP-encapsulated RNA can be manufactured by rapidly mixing an RNA solution (e.g., RNA product solution) described herein with a lipid formulation (comprising, for example, at least one cationic lipid and optionally one or more other lipid components in an organic solvent) described herein under conditions such that a sharp change in the solubility of the one or more lipid components is triggered, which drives the lipid to self-assemble in LNP form. In some embodiments, suitable buffers comprise tris, histidine, citrate, acetate, phosphate and/or succinate. In some embodiments, 1, 2, 3, or more of the foregoing buffers are excluded. The pH of the liquid formulation is related to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidified buffer may be at least half pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some embodiments, the properties of the cationic lipid are selected such that initial formation of the particle occurs by binding to an oppositely charged backbone of a nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which in some embodiments, for example, can result in an encapsulation efficiency that is much higher than that achieved if there were no interaction between the nucleic acid and at least one of the lipid components. In certain embodiments, the nucleic acid, when present in the lipid nanoparticle, is resistant to nuclease degradation in aqueous solution.

Lipid nanoparticles comprising nucleic acids and methods of making the same are disclosed, for example, in U.S. patent publication nos. 2004/0142025, 2007/0042031, and PCT publication nos. WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

Some embodiments described herein relate to compositions, methods, and uses of more than one, e.g., 2, 3, 4, 5, 6, or even more, nucleic acid species (such as RNA species). In LNP formulations, it is possible to formulate each nucleic acid species separately into individual LNP formulations. In this case, each individual LNP formulation will comprise one nucleic acid species. The individual LNP formulations may exist in separate entities, for example in separate containers. Such formulations may be obtained by separately providing each nucleic acid species (typically each in the form of a nucleic acid-containing solution) and suitable cationic or cationizable lipid or lipid-like material and cationic polymer that allow for the formation of LNP. The individual particles will contain only the specific nucleic acid species provided when the particles are formed (individual particle formulations).

In some embodiments, a composition, such as a pharmaceutical composition, comprises more than one individual LNP formulation. The respective pharmaceutical compositions are referred to as mixed LNP formulations. The mixed LNP formulation according to the present invention may be obtained from the steps of separately forming individual LNP formulations as described above, followed by mixing the individual LNP formulations. From the step of mixing, a formulation comprising a mixed population of LNPs containing nucleic acids can be obtained. Individual populations of LNPs may be together in one container, comprising a mixed population of individual LNP formulations.

Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations may be obtained from a combination formulation (typically a combination solution) that provides different RNA species, suitable cationic or cationizable lipid or lipid-like materials and cationic polymers that allow for the formation of LNP. In contrast to the mixed LNP formulation, the combined LNP formulation will typically comprise LNP containing more than one RNA species. In a combined LNP composition, the different RNA species are typically present together in a single particle.

[ A ] cationic polymeric Material ]

In view of the high chemical flexibility of polymeric materials, polymeric materials are typically used for nanoparticle-based delivery. Typically, negatively charged nucleic acids are electrostatically aggregated into nanoparticles using cationic materials. These positively charged groups typically consist of amines whose protonation state changes in the pH range between 5.5 and 7.5, which is thought to cause ion imbalance leading to endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethylenimine, as well as naturally occurring polymers such as chitosan, have all been applied for nucleic acid delivery and are suitable as cationic materials suitable for use in some embodiments herein. In addition, some researchers have synthesized polymeric materials that are specific for nucleic acid delivery. In particular, poly (P-amino esters) are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

As used herein, "polymeric material" has its ordinary meaning, such as a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some embodiments, such repeating units may all be uniform, or in some cases, more than one type of repeating unit may be present within the polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer, such as a protein. In some cases, additional moieties may also be present in the polymeric material, for example targeting moieties, such as those described herein.

Those skilled in the art will appreciate that when more than one type of repeating unit is present in a polymer (or polymeric moiety), then the polymer (or polymeric moiety) is referred to as a "copolymer". In some embodiments, the polymer (or polymeric moiety) used according to the present invention may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, in some embodiments, the repeating units may be arranged in a random order, or alternatively, in some embodiments, the repeating units may be arranged in an alternating order, or as a "block" copolymer, for example, comprising one or more regions each comprising a first repeating unit (e.g., a first block), one or more regions each comprising a second repeating unit (e.g., a second block), and the like. The block copolymer may have two (diblock copolymer), three (triblock copolymer) or a greater number of different blocks.

In certain embodiments, the polymeric materials used in accordance with the present invention are biocompatible. Biocompatible materials are materials that do not generally lead to significant cell death at moderate concentrations. In certain embodiments, the biocompatible material is biodegradable, e.g., capable of being chemically and/or biologically degraded within a physiological environment, such as within the body. In certain embodiments, the polymeric material may be or comprise protamine or a polyalkyleneimine, in particular protamine.

It will be appreciated by those skilled in the art that the term "protamine" is often used to refer to any of a variety of strongly basic proteins that have a relatively low molecular weight, are rich in arginine, and are found to bind, inter alia, DNA, but not to the somatic histones, in sperm cells of various animals (e.g., fish). In particular, the term "protamine" is commonly used to refer to proteins that are present in fish sperm that are strongly alkaline, soluble in water, do not solidify upon heating, and produce primarily arginine upon hydrolysis. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

In some embodiments, the term "protamine" as used herein refers to a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and/or multimeric forms of the amino acid sequence or fragments thereof, as well as (synthetic) polypeptides that are artificial and specifically designed for a specific purpose and that cannot be isolated from a natural or biological source.

In some embodiments, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine. In some embodiments, the polyalkyleneimine is Polyethyleneimine (PEI). In some embodiments, the polyalkyleneimine is a linear polyalkyleneimine, such as a linear Polyethyleneimine (PEI).

Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those cationic materials capable of electrostatically binding nucleic acids. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric material that can bind to a nucleic acid, for example, by forming a complex with the nucleic acid or forming vesicles that enclose or encapsulate the nucleic acid.

In some embodiments, the particles described herein may comprise polymers other than cationic polymers, such as non-cationic polymeric materials and/or anionic polymeric materials. Anionic and neutral polymeric materials are collectively referred to herein as non-cationic polymeric materials.

[ B ] lipid and lipid-like Material ]

The terms "lipid" and "lipid-like material" are used herein to refer to molecules comprising one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. According to the present invention, the lipids and lipid-like materials may be cationic, anionic or neutral. At the selected pH, the neutral lipid or lipid-like material exists in an uncharged or neutral zwitterionic form.

The term "lipid" refers to a group of organic compounds characterized as insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight classes, fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids, and sterol-containing metabolites (such as cholesterol) and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosyl glycerols (glycosylglycerol) and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramide sphingomyelin (phosphosphingolipid) (e.g., sphingomyelin (sphingomyelin), phosphorylcholine), and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and derivatives thereof, and tocopherols and derivatives thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the lipids may be excluded from the LNP of the invention.

The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" refers to a substance that is structurally and/or functionally related to a lipid, but may not be considered a lipid in a strict sense. For example, the term includes compounds that are capable of forming an amphiphilic layer when present in vesicles, multilamellar/unilamellar liposomes or membranes in an aqueous environment, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. In general, the term refers to molecules comprising hydrophilic and hydrophobic portions of different structural tissues, which may or may not be lipid-like.

In some embodiments, the RNA solution and lipid formulation mixture or composition thereof may comprise cationic lipids, neutral lipids, cholesterol, and/or polymer (e.g., polyethylene glycol) conjugated lipids, which form lipid nanoparticles surrounding the RNA molecule. Thus, in some embodiments, the LNP can comprise a cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer-bound lipids (e.g., PEG-lipids), or a combination thereof. In some embodiments, 1, 2, 3 or more of the foregoing excipients may be excluded from the LNP of the invention. In some embodiments, the lipid is present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent (e.g., an RNA molecule) for treating a particular disease or condition of interest (e.g., those associated with RSV). In some embodiments, the LNP comprises or encapsulates a nucleic acid molecule.

[ I ] cationic lipid ]

Cationic or cationizable lipid or lipid-like material refers to a lipid or lipid-like material capable of being positively charged and capable of electrostatically binding nucleic acids. As used herein, "cationic lipid" or "cationic lipid-like material" refers to a lipid or lipid-like material having a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acids by electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries a positive charge. Exemplary cationic lipids include one or more amino groups bearing a positive charge. Cationic lipids can encapsulate negatively charged RNAs.

In some embodiments, the cationic lipid is ionizable such that it may exist in either a positively charged or neutral form, depending on the pH. Ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, it is believed that this ionizable behavior promotes efficacy by helping endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH. For the purposes of the present invention, the term "cationic lipid" or "cationic lipid-like material" encompasses such "cationically ionizable" lipids or lipid-like materials, unless contradicted by context.

In some embodiments, the cationic lipid may comprise or comprise about 10mol% to about 100mol%, about 20mol% to about 100mol%, about 30mol% to about 100mol%, about 40mol% to about 100mol%, or about 50mol% to about 100mol% of the total lipid present in the particle. In some embodiments, the cationic lipid may or may not be at least, up to, just below, or in between (inclusive or exclusive) 10mol%, 20mol%, 30mol%, 40mol%, 50mol%, 60mol%, 70mol%, 80mol%, 90mol%, or 100mol% of the total lipid present in the particle, or any range or value derivable therein.

Examples of cationic lipids include, but are not limited to, ((4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate), 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP), N-dimethyl-2, 3-dioleoyloxypropylamine (DODMA), 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA), 3- (N- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol), dimethyl Dioctadecyl Ammonium (DDAB), 1, 2-dioleoyl-3-dimethylammonium-propane (DODAP), 1, 2-Diacyloxy-3-dimethylammonium propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyl-dimethylammonium chloride (DODAC), 1, 2-distearyloxy-N, N-dimethyl-3-aminopropane (DSDMA), 2, 3-di (tetradecyloxy) propyl- (2-hydroxyethyl) -dimethylammonium nitrogen (DMRIE), 1, 2-dimyristoyl-sn-glycero-3-ethylphosphoric acid choline (DMEPC), 1, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 2, 3-Dioleoyloxy-N- [2 (argininamide) ethyl ] -N, N-dimethyl-1-propanamide onium (propanamium) trifluoroacetate (DOSPA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLenDMA), dioctadecyl amidoglycyl spermine (DOGS), 3-dimethylamino-2- (cholesterol-5-en-3-beta-oxybutynin-4-oxy) -1- (cis, cis-9, 12-octadecadienyloxy) propane (CLinDMA), 2- [5'- (cholest-5-en-3-beta-oxy) -3' -oxapentenyloxy) -3-dimethyl-1- (cis, cis-9 ',12' -octadecadienyloxy) propane (CpLinDMA), N-dimethyl-3, 4-dioleyloxybenzyl amine (DMOBA), 1,2-N, N '-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2, 3-dioleoyloxy-N, N-dimethylpropylamine (DLinDAP), 1,2-N, N' -dioleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1, 2-Dioleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP), 2-diiodo-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2-diiodo-4-dimethylaminoethyl- [1,3] -dioxolane (DLin-K-XTC 2-DMA), 2-diiodo-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), thirty-seven carbon-6,9,28,31-tetraen-19-yl-4- (dimethylamino) butanoate (DLin-MC 3-DMA), N- (2-hydroxyethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propylamine bromide (DMRIE), (+ -) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (cis-9-tetradecenyloxy) -1-propylamine bromide (GAP-DMRIE), (+ -) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (dodecyloxy) -1-propylamine bromide (GAP-DLRIE), (+ -) -N- (3-aminopropyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propylamine bromide (GAP-DMRIE), and, n- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) -1-propylamine (bAE-DMRIE), N- (4-carboxybenzyl) -N, N-dimethyl-2, 3-bis (oleoyloxy) propan-1-ammonium bromide (DOBAQ), 2- ({ 8- [ (3 b) -cholest-5-en-3-yloxy ] Octyl } oxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (Octyl-CLinDMA), 1, 2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1, 2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-amino-propyl) amino ] butylcarboxamido) ethyl ] -3, 4-bis [ oleoyl ] -benzamide (MVL 5), 1, 2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2, 3-bis (dodecyloxy) -N- (2-hydroxyethyl) -N, N-dimethylpropan-1-ammonium bromide (DLRIE), N- (2-aminoethyl) -N, N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-ammonium bromide (DMORE), 8,8' - ((((2 (dimethylamino) ethyl) thio) carbonyl) aminoidene) dioctanoic acid di ((Z) -non-2-en-1-yl ester) (ATX), N-dimethyl-2, 3-bis (dodecyloxy) propan-1-amine (DLDMA), N-dimethyl-2, 3-bis (tetradecyloxy) propan-1-amine (DMDMA), di ((Z) -non-2-en-1-yl) -9- ((4- (dimethylaminobutyryl) oxy) heptadecanedioic acid ester (L319), N-dodecyl-3- ((2-dodecylcarbamoyl-ethyl) - {2- [ (2-dodecylcarbamoyl-ethyl) -2- { (2-dodecylcarbamoyl-ethyl) - [2- (2-dodecylcarbamoyl-ethylamino) -ethyl ] -amino } -ethylamino) propanamide (lipid 98N 12-5), 1- [2- [ bis (2-hydroxydodecyl) amino ] ethyl- [2- [4- [2- [ bis (2 hydroxydodecyl) amino ] ethyl ] piperazin-1-yl ] ethyl ] amino ] dodec-n-2-ol (lipid 02-200), C12-200, or 8- ((2-hydroxyethyl) (6-oxo-6- (undecoxy) hexyl) amino) heptadec-9-yl octanoate (SM-102). in some embodiments, 1,2, 3, 4, 5 or more of the foregoing cationic lipids may be excluded from the LNP of the invention.

In some embodiments, the ionizable cationic lipids of the invention comprise a compound of formula (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ' or-R ' M ' R ';

R ₂ and R ₃ are independently H, C _1～14 alkyl, C _2～14 alkenyl, -R xr ", -YR", OR-R OR ", and/OR R ₂ and R ₃ together with the atoms to which they are attached form a heterocycle OR carbocycle;

R ₄ is a C _3～6 carbocycle, - (CH ₂)_nQ、-(CH₂)_nCHQR、-CHQR、-CQ(R)₂) OR unsubstituted C _1-6 alkyl, wherein Q is a carbocycle, heterocycle 、-OR、-O(CH₂)_nN(R)₂、-C(O)OR、-OC(O)R、-CX₃、-CX₂H、-CXH₂、-CN、-N(R)₂、-C(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)C(O)N(R)₂、-N(R)C(S)N(R)₂、-N(R)R₈、-O(CH₂)_nOR、-N(R)C(＝NR₉)N(R)₂、-N(R)C(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)₂R、-N(OR)C(O)OR、-N(OR)C(O)N(R)₂、-N(OR)C(S)N(R)₂、-N(OR)C(＝NR₉)N(R)₂、-N(OR)C(＝CHR₉)N(R)₂、-C(＝NR₉)N(R)₂、-C(＝NR₉)R、-C(O)N(R)OR OR-C (R) N (R) ₂ C (O) OR, and/OR each N is independently 1, 2, 3, 4 OR 5;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

M and M' are independently -C(O)O-、-OC(O)-、-C(O)N(R′)-、-N(R′)C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)₂-、-S-S-、 aryl or heteroaryl;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

r ₈ is a C _3～6 carbocycle or heterocycle;

R ₉ is H, CN, NO ₂、C_1～6 alkyl, -OR, -S (O) ₂R、-S(O)₂N(R)₂、C_2～6 alkenyl, C _3～6 carbocycle OR heterocycle;

Each R is C _1～3 alkyl, C _2～3 alkenyl, or H;

Each R ' is C1-18 alkyl, C _2～18 alkenyl, -R x YR ', -YR ' or H;

Each R "is C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _2～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

M is 5, 6, 7, 8, 9, 10, 11, 12 or 13.

In some embodiments, a subset of compounds of formula (I) include those wherein when R ₄ is- (CH ₂)_nQ、-(CH₂)_n CHQR, -CHQR or-CQ (R) ₂ then (I) when N is 1, 2,3, 4 or 5, Q is not-N (R) ₂, or (ii) when N is 1 or 2, Q is not a 5,6 or 7 membered heterocycloalkyl.

In some embodiments, another subset of compounds of formula (I) include those wherein R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ', or-R ' M ' R ';

R ₄ is a C _3～6 carbocycle, - (CH ₂)_nQ、-(CH₂)_nCHQR、-CHQR、-CQ(R)₂) or unsubstituted C _1～6 alkyl, wherein Q is a C _3～6 carbocycle, a 5-to 14-membered heteroaryl 、-OR、-O(CH₂)_nN(R)₂、-C(O)OR、-OC(O)R、-CX₃、-CX₂H、-CXH₂、-CN、-C(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)C(O)N(R)₂、-N(R)C(S)N(R)₂、-CRN(R)₂C(O)OR、-N(R)R₈、-O(CH₂)_nOR、-N(R)C(＝NR₉)N(R)₂、-N(R)C(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)₂R、-N(OR)C(O)OR、-N(OR)C(O)N(R)₂、-N(OR)C(S)N(R)₂、-N(OR)C(＝NR₉)N(R)₂、-N(OR)C(＝CHR₉)N(R)₂、-C(＝NR₉)N(R)₂、-C(＝NR₉)R、-C(O)N(R)OR having one or more heteroatoms comprising N, O or S, or a 5-to 14-membered heterocycloalkyl having one or more heteroatoms comprising N, O and S, substituted with one or more substituents comprising oxo (=O), OH, amino, mono-or dialkylamino, or C _1～3 alkyl, and/or each n is independently 1, 2, 3, 4, or 5;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

r ₈ is a C _3～6 carbocycle or heterocycle;

each R is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R' is independently C _1～18 alkyl, C _2～18 alkenyl, -R x YR ", -YR", or H;

Each R "is independently C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _2～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

M is 5, 6, 7, 8, 9, 10, 11, 12 or 13, and/or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

In some embodiments, another subset of compounds of formula (I) include those wherein:

R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ' or-R ' M ' R ';

R ₂ and R ₃ are independently H, C _1-14 alkyl, C _2-14 alkenyl, -R xr ", -YR", OR-R OR ", and/OR R ₂ and R ₃ together with the atoms to which they are attached form a heterocycle OR carbocycle;

R ₄ is a C _3～6 carbocycle, - (CH ₂)_nQ、-(CH₂)_nCHQR、-CHQR、-CQ(R)₂) or unsubstituted C _1-6 alkyl wherein Q is a C _3～6 carbocycle, a 5-to 14-membered heterocycle 、-OR、-O(CH₂)_nN(R)₂、-C(O)OR、-OC(O)R、-CX₃、-CX₂H、-CXH₂、-CN、-C(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)C(O)N(R)₂、-N(R)C(S)N(R)₂、-CRN(R)₂C(O)OR、-N(R)R₈、-O(CH₂)_nOR、-N(R)C(＝NR₉)N(R)₂、-N(R)C(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)₂R、-N(OR)C(O)OR、-N(OR)C(O)N(R)₂、-N(OR)C(S)N(R)₂、-N(OR)C(＝NR₉)N(R)₂、-N(OR)C(＝CHR₉)N(R)₂、-C(＝NR₉)R、-C(O)N(R)OR having one or more heteroatoms comprising N, O or S, or-C (=NR ₉)N(R)₂, and/or each n is independently 1,2,3, 4 or 5, and/or when Q is a 5-to 14-membered heterocycle and (i) R ₄ is- (CH ₂)_n Q wherein n is 1 or 2, or (ii) R ₄ is- (CH ₂)_n CHQR wherein n is 1, or (iii) R ₄ is-CHQR and-CQ (R) ₂, then Q is a 5-to 14-membered heteroaryl or 8-to 14-membered heterocycloalkyl;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

M and M' are independently -C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)₂-、-S-S-、 aryl or heteroaryl;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

r ₈ is a C _3～6 carbocycle or heterocycle;

R ₉ is H, CN, NO ₂、C_1～6 alkyl, -OR, -S (O) ₂R、-S(O)₂N(R)₂、C_2-6 alkenyl, C _3-6 carbocycle OR heterocycle;

each R is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

Each R "is independently C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _2～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ' or-R ' M ' R ';

R ₄ is a C _3～6 carbocycle, - (CH ₂)_nQ、-(CH₂)_nCHQR、-CHQR、-CQ(R)₂) or unsubstituted C _1～6 alkyl, wherein Q is a C _3～6 carbocycle, a 5-to 14-membered heteroaryl 、-OR、-O(CH₂)_nN(R)₂、-C(O)OR、-OC(O)R、-CX₃、-CX₂H、-CXH₂、-CN、-C(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)C(O)N(R)₂、-N(R)C(S)N(R)₂、-CRN(R)₂C(O)OR、-N(R)R₈、-O(CH₂)_nOR、-N(R)C(＝NR₉)N(R)₂、-N(R)C(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)₂R、-N(OR)C(O)OR、-N(OR)C(O)N(R)₂、-N(OR)C(S)N(R)₂、-N(OR)C(＝NR₉)N(R)₂、-N(OR)C(＝CHR₉)N(R)₂、-C(＝NR₉)R、-C(O)N(R)OR having one or more heteroatoms comprising N, O or S, or-C (=nr ₉)N(R)₂, and/or each n is independently 1, 2, 3, 4, or 5;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

r ₈ is a C _3～6 carbocycle or heterocycle;

each R is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

Each R "is independently C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _2～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ' or-R ' M ' R ';

R ₂ and R ₃ are independently H, C _2～14 alkyl, C _2～14 alkenyl, -R xr ", -YR", OR-R OR ", and/OR R ₂ and R ₃ together with the atoms to which they are attached form a heterocycle OR carbocycle;

R ₄ is- (CH ₂)_n Q or- (CH ₂)_n CHQR) wherein Q is-N (R) ₂ and/or N is 3,4 or 5;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

each R is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

Each R "is independently C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _1～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

R ₁ is C _5～30 alkyl, C _5～20 alkenyl, -R x YR ', -YR ' or-R ' M ' R ';

R ₂ and R ₃ are independently C _1～14 alkyl, C _2～14 alkenyl, -R YR ', -YR ' OR-R OR ', and/OR R ₂ and R ₃ together with the atoms to which they are attached form a heterocycle OR carbocycle;

R ₄ is- (CH ₂)_nQ、-(CH₂)_n CHQR, -CHQR or-CQ (R) ₂, wherein Q is-N (R) ₂, and/or N is 1,2,3, 4, or 5;

Each R ₅ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

each R ₆ is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

M and M' are independently -C(O)O-、-OC(O)-、-C(O)N(R′)-、-N(R′)C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR′)O-、-S(O)₂-、-S-S-、 aryl or heteroaryl;

r ₇ is C _1～3 alkyl, C _2～3 alkenyl or H;

each R is independently C _1～3 alkyl, C _2～3 alkenyl, or H;

Each R "is independently C _3～14 alkyl or C _3～14 alkenyl;

Each R is independently C _1～12 alkyl or C _1～12 alkenyl;

each Y is independently a C _3～6 carbocycle;

each X is independently F, cl, br or I, and

In some embodiments, a subset of compounds of formula (I) include those of formula (IA):

OR a pharmaceutically acceptable salt, tautomer, prodrug OR stereoisomer thereof, wherein I is 1, 2, 3,4 OR 5;m is 5, 6,7,8 OR 9;M ₁ is a bond OR M '; R ₄ is unsubstituted C _1～3 alkyl OR- (CH ₂)_n Q, wherein Q is OH、-NHC(S)N(R)₂、-NHC(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)R₈、-NHC(＝NR₉)N(R)₂、-NHC(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、 heteroaryl OR heterocycloalkyl; M and M' are independently-C (O) O-, -OC (O) -, -C (O) N (R ') -, -P (O) (OR') O-, -S-, aryl OR heteroaryl; and R ₂ and R ₃ are independently H, C _1～14 alkyl OR C _2～14 alkenyl.

In some embodiments, a subset of compounds of formula (I) include those of formula (II):

OR a pharmaceutically acceptable salt, tautomer, prodrug OR stereoisomer thereof, wherein I is 1,2, 3, 4 OR 5;M ₁ is a bond OR M '; R ₄ is unsubstituted C _1～3 alkyl OR- (CH ₂)_n Q, wherein N is 2,3 OR 4 and Q is OH、-NHC(S)N(R)₂、-NHC(O)N(R)₂、-N(R)C(O)R、-N(R)S(O)₂R、-N(R)R₈、-NHC(＝NR₉)N(R)₂、-NHC(＝CHR₉)N(R)₂、-OC(O)N(R)₂、-N(R)C(O)OR、 heteroaryl OR heterocycloalkyl; M and M' are independently-C (O) O-, -OC (O) -, -C (O) N (R ') -, P (O) (OR') O-, -S-, aryl OR heteroaryl, and R ₂ and R ₃ are independently H, C _1～14 alkyl OR C _2～14 alkenyl, in some embodiments, a subset of compounds of formula (I) include those of formula (Ila), (iib), (lIc) OR (lIe).

Or a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof, wherein R ₄ is as described herein.

In some embodiments, a subset of compounds of formula (I) include those of formula (IId):

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein n is 2,3 or 4, and m, R', R ", and R ₂～R₆ are as described herein. For example, each of R ₂ and R ₃ may independently be C _5～14 alkyl or C _5～14 alkenyl.

In some embodiments, the ionizable cationic lipids of the invention comprise a compound having the structure:

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 is a direct bond;

Each of G ¹ and G ² is independently unsubstituted C ₁～C₁₂ alkylene or C ₁～C₁₂ alkenylene, G is C ₁～C₂₄ alkylene, C ₁～C₂₄ alkenylene, C ₃～C₈ cycloalkylene, 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;

R ⁴ is C ₁～C₁₂ alkyl;

r ⁵ is H or C ₁～C₆ alkyl

X is 0, 1 or 2.

In some of the foregoing embodiments, the ionizable cationic lipid comprises a compound having one of the following structures:

a is 3-8 membered cycloalkyl or cycloalkylene ring;

R ⁶ is, independently at each occurrence, H, OH or C ₁～C₂₄ alkyl, and

N is an integer ranging from 1 to 15.

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein y and z are each independently integers in the range of 1 to 12.

In any of the foregoing embodiments, one of L ¹ or L ² is-OCCO) -. For example, in some embodiments, each of L ¹ and L ² is-O (c=o) -. In some embodiments of any of the foregoing, L ¹ and L ² are each independently- (c=o) O-or-O (c=o) -. For example, in some embodiments, each of L ¹ and L ² is- (c=o) O-.

Or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

In some of the foregoing embodiments, n is an integer in the range of 2 to 12, e.g., 2 to 8 or 2 to 4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some of the foregoing embodiments, y and z are each independently integers in the range of 2 to 10. For example, in some embodiments, y and z are each independently integers in the range of 4-9 or 4-6.

In some of the foregoing embodiments, R ⁶ is H. In other of the foregoing embodiments, R ⁶ is C ₁～C₂₄ alkyl. In other embodiments, R ⁶ is OH. In some embodiments, G is unsubstituted. In other embodiments, G ³ is substituted. In various embodiments, G ³ is a linear C ₁～C₂₄ alkylene or linear C ₁～C₂₄ alkenylene.

In some other of the foregoing embodiments, R ¹ or R ², or both, are C ₆～C₂₄ alkenyl. For example, in some embodiments, R ¹ and R ² each independently have the following structure:

Wherein:

R ^7a and R ^7b are independently at each occurrence H or C ₁～C₁₂ alkyl, and a is an integer from 2 to 12,

Wherein R ^7a、R^7b and a are each selected such that R ¹ and R ² each independently comprise 6 to 20 carbon atoms. For example, in some embodiments, a is an integer in the range of 5-9 or 8-12.

In some of the foregoing embodiments, at least one occurrence of R ^7a is H. For example, in some embodiments, R ^7a is H at each occurrence. In other various embodiments of the foregoing, at least one occurrence of R ^7b is C ₁～C₈ alkyl. For example, in some embodiments, the C ₁～C₈ alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

In various embodiments, R ¹ or R ², or both, have one of the following:

In some of the foregoing embodiments, R is OH, CN, -C (=o) OR ⁴-OC(＝O)R⁴, OR-NHC (=o) R ⁴. In some embodiments, R ⁴ is methyl or ethyl.

It is to be understood that any of the embodiments of the compounds set forth above and any particular substituents and/or variables in the compounds set forth above may be independently combined with other embodiments and/or substituents and/or variables of the compounds to form embodiments of the invention not specifically set forth above. In addition, where a list of substituents and/or variables is recited for any particular substituent and/or variable in a particular embodiment and/or technical scheme, it is to be understood that each individual substituent and/or variable may be absent from the particular embodiment and/or technical scheme, and that the list of residues of substituents and/or variables is to be considered within the scope of the present invention. It is understood that in this specification, combinations of substituents and/or variables of the depicted formulas are permissible only if such actions result in stable compounds.

In some embodiments, the cationic lipid is

In some embodiments, the lipid nanoparticle comprises one or more cationic lipids. In one embodiment, the lipid nanoparticle comprises (4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (ALC-0315) having the formula:

exemplary cationic lipids are disclosed, for example, in U.S.10,166,298, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Representative cationic lipids include:

in some embodiments, 1,2, 3, 4, 5 or more of the foregoing cationic lipids may be excluded from the LNP of the invention.

In some embodiments, the RNA-LNP comprises a cationic lipid, an RNA molecule described herein, and one or more of a neutral lipid, a steroid, a pegylated lipid, or a combination thereof. If more than one cationic lipid is incorporated into the LNP, these percentages apply to the combined cationic lipids. In one embodiment, the cationic lipid is present in the LNP in an amount such as at least, up to, just below, or between (inclusive or exclusive) or about below, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole% (mol%). In some embodiments, two or more cationic lipids are incorporated into the LNP. If more than one cationic lipid is incorporated into the LNP, the foregoing percentages apply to the incorporated cationic lipids.

In some embodiments of the invention, the LNP comprises a combination or mixture of any of the lipids described above.

[ Ii ] Polymer-bound lipids ]

In some embodiments, the LNP comprises a polymer-bound lipid. The term "polymer-bound lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. Examples of polymer-bound lipids are pegylated lipids (e.g., polyethylene glycol-lipids, PEG-lipids). In certain embodiments, the LNP comprises an additional stabilizing lipid that is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid moiety and a polyethylene glycol moiety.

Pegylated lipids are known in the art and include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC or PEG-CerC), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2- [ (polyethylene glycol) -2000] -N, N-ditetradecyl) acetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s-DMG (1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoyl glycerol). In one embodiment, the polyethylene glycol-lipid is N- [ (methoxypolyethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises a pegylated diacylglycerol (PEG-DAG), such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinic diacylglycerol (PEG-S-DAG), such as 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- ((ω -methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropyl carbamate, such as a co-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate or a2, 3-di (tetradecyloxy) propyl-N- (ω -methoxy (polyethoxy) ethyl) carbamate, the entire disclosure of which is disclosed in, for example, US 9,737,619, incorporated herein by reference in its entirety.

In some embodiments, the composition comprises a pegylated lipid having the structure:

Or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

R ⁸ and R ⁹ are each independently a straight or branched chain containing 10 to 30 carbon atoms, a saturated or unsaturated alkyl chain, wherein the alkyl chain is optionally interrupted by one or more ester bonds, and w has an average value in the range of 30 to 60. In some embodiments, R ⁸ and R ⁹ are each independently a straight saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43-53. In other embodiments, the average w is at or about 45. In various other embodiments, the average w is at or about 49.

In some embodiments, the lipid nanoparticle comprises a polymer-bound lipid. In one embodiment, the lipid nanoparticle comprises 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159) having the formula:

In various embodiments, the molar ratio of cationic lipid to pegylated lipid is in the range of or about 100:1 to about 20:1, such as 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, or any range or value derivable therein.

In certain embodiments, the PEG-lipid is or is not present in the LNP in an amount of, or about 1 to about 10 mole% (mol%) (e.g., at least, up to, just below, or between (inclusive or exclusive) 1,2, 3, 4, 5,6, 7, 8, 9, or 10 mol%) relative to the total lipid content of the nanoparticle.

In some embodiments, the ratio of PEG in the lipid nanoparticle formulation may be increased or decreased, and/or the carbon chain length of the PEG lipid may be modified to alter the pharmacokinetics and/or biodistribution of the lipid nanoparticle formulation.

[ Iii ] additional lipids ]

In certain embodiments, the LNP comprises one or more additional lipids or lipid-like materials that stabilize the particle during its formation. Suitable stabilizing or structuring lipids include non-cationic lipids, such as neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNP by adding other hydrophobic moieties, such as cholesterol and lipids other than ionizable/cationic lipids or lipid-like materials, can enhance particle stability and nucleic acid delivery efficacy.

As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. In some embodiments, the additional lipid comprises one of (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydro-sphingomyelin, cephalin, and cerebrosides. Exemplary phospholipids include, for example, phosphatidyl choline, e.g., diacyl phosphatidyl choline, such as distearoyl phosphatidyl choline (DSPC), dioleoyl phosphatidyl choline (DOPC), dimyristoyl phosphatidyl choline (DMPC), eicosanoyl phosphatidyl choline, dilauroyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl choline (DPPC), dioleoyl phosphatidyl glycerol (DOPG), dipalmitoyl phosphatidyl glycerol (DPPG), ditolyl phosphatidyl choline (DAPC), dibisbehenyl phosphatidyl choline (DBPC), di (tricosyl) phosphatidyl choline (DTPC), and di (tricosyl) phosphatidyl choline (DTPC), Di (tetracosyl) phosphatidylcholine (DLPC), palmitoyl oleoyl-phosphatidylcholine (POPC), 1, 2-di-O-octadecenyl-sn-glycero-3-phosphorylcholine (18:0 DietherPC), 1-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphorylcholine (OChemsPC) and 1-hexadecyl-sn-glycero-3-phosphorylcholine (C16 Lyso PC), and phosphatidylethanolamine, for example diacylphosphatidylethanolamine, such as dioleoyl-phosphatidylethanolamine (DOPE), 1, 2-di-undecanoyl-sn-glycero-phosphorylcholine (DUPC), Palmitoyl Oleoyl Phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), 1-phytoyl-phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), 1, 2-di-trans-oleoyl-sn-glycero-3-phosphate ethanolamine (trans DOPE), 1, 2-di-linolenoyl-sn-glycero-3-phosphate choline, 1, 2-di-arachidonoyl-sn-glycero-3-phosphate choline, 1, 2-di-docosahexaenoic acid-sn-glycero-3-phosphate choline, 1, 2-di-phytanoyl-sn-glycero-3-phosphate ethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphate ethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-arachidonoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-docosahexaenoic acyl-sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycero) sodium salt (DOPG), sphingomyelin, and mixtures thereof. In some embodiments, 1,2, 3, 4, 5 or more of the foregoing neutral lipids may be excluded from the LNP of the invention.

In one embodiment, the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) having the formula:

In some embodiments, the LNP comprises neutral lipids, and the neutral lipids comprise one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and/or SM. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing neutral lipids may be excluded from the LNP of the invention.

In various embodiments, the LNP further comprises a steroid or steroid analog. "steroid" is a compound comprising the following carbon skeleton:

In certain embodiments, the steroid or steroid analog is cholesterol, fecal sterol, plant sterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomato secondary alkali, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing steroids or steroid analogs may be excluded from the LNP of the invention. In certain embodiments, the steroid or steroid analog is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, stigmasterol, cholesteryl-2 '-hydroxyethyl ether, cholesteryl-4' -hydroxybutyl ether, tocopherols, and derivatives thereof, and mixtures thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing cholesterol derivatives may be excluded from the LNP of the invention. In one embodiment, the cholesterol has the formula:

Without being bound by any theory, the amount of at least one cationic lipid can affect important nucleic acid particle characteristics, such as charge, particle size, stability, tissue selectivity, and biological activity of the nucleic acid, as compared to the amount of at least one additional lipid. Thus, in some embodiments, the molar ratio of cationic lipid to neutral lipid is in the range of or about 2:1 to about 8:1, or in the range of or about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

In some embodiments, the non-cationic lipid (e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol)) may comprise total lipid present in the particle at or at about 0mol% to about 90mol%, at or at about 0mol% to about 80mol%, at or at about 0mol% to about 70mol%, at or at about 0mol% to about 60mol%, or at about 0mol% to about 50 mol%. In some embodiments, the non-cationic lipid (e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol)) may or may not be at least, up to, just below, or in between (inclusive or exclusive) 0mol%, 10mol%, 20mol%, 30mol%, 40mol%, 50mol%, 60mol%, 70mol%, 80mol%, or 90mol% of the total lipid present in the particle.

Characterization and analysis of RNA molecules

The RNA molecules described herein can be analyzed and characterized using various methods. Analysis may be performed before and/or after capping. Alternatively, the analysis may be performed before and/or after affinity purification based on multi-a capture. In another embodiment, the analysis may be performed before and/or after additional purification steps, such as anion exchange chromatography and the like. For example, RNA template quality can be determined using an electrophoresis system based on a bioanalyzer chip. In other embodiments, RNA template purity is analyzed using analytical reverse phase HPLC. Capping efficiency can be analyzed using, for example, total nuclease digestion followed by MS/MS quantification of dinucleotide cap species relative to uncapped GTP species. In vitro efficacy can be analyzed by, for example, transfection of RNA molecules into human cell lines. Protein expression of the polypeptide of interest can be quantified using methods such as ELISA and/or flow cytometry techniques. Immunogenicity can be analyzed, for example, by transfection of RNA molecules into cell lines (e.g., PBMCs) that are indicative of innate immune stimulation. Cytokine induction can be analyzed using, for example, methods such as ELISA to quantify cytokines, e.g., interferon- α. Biodistribution can be analyzed by, for example, bioluminescence measurement. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing analytical methods may be excluded.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when assessed in an organism to which a composition or pharmaceutical formulation comprising the RNA polynucleotide is administered, an increase in expression of the gene of interest (e.g., antigen), an increase in expression duration (e.g., prolonged expression) of the gene of interest (e.g., antigen), an increase in expression of the gene of interest (e.g., antigen) and an increase in expression duration (e.g., prolonged expression), a decrease in the interaction with IFIT1 of the RNA polynucleotide, and/or an increase in translation of the RNA polynucleotide is observed relative to an appropriate reference. In some embodiments, 1,2,3, 4, 5 or more of the foregoing features may not be observed after administration of a composition or pharmaceutical formulation comprising an RNA molecule of the invention.

In some embodiments, the reference comprises administering an RNA polynucleotide that is otherwise similar but does not have an m7 (3 'ome g) (5') ppp (5 ') (2' ome ai) pG2 cap. In some embodiments, reference is made to an organism comprising an RNA polynucleotide administered that is otherwise similar but does not have the cap proximal sequences disclosed herein. In some embodiments, the reference comprises administering an organism that is otherwise similar but has an RNA polynucleotide that hybridizes to the sequence.

In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression is determined at least 24 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, increased expression is determined at least 48 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression is determined at least 72 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression is determined at least 96 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression is determined at least 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide.

In some embodiments, elevated expression is measured at or about 24-120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, increased expression is measured 24-110 hours, 24-100 hours, 24-90 hours, 24-80 hours, 24-70 hours, 24-60 hours, 24-50 hours, 24-40 hours, 24-30 hours, 30-120 hours, 40-120 hours, 50-120 hours, 60-120 hours, 70-120 hours, 80-120 hours, 90-120 hours, 100-120 hours, or 110-120 hours after administration of a composition or pharmaceutical preparation comprising an RNA polynucleotide.

In some embodiments, the expression of the gene of interest (e.g., antigen) is increased or not increased by at least 2-fold to at least 10-fold. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least a factor of 2. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least a factor of 3. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least a factor of 4. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least 6-fold. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least 8-fold. In some embodiments, expression of a gene of interest (e.g., an antigen) is increased by at least a factor of 10.

In some embodiments, the expression of the gene of interest (e.g., antigen) is increased or increased by a factor of about 2 to about 50. In some embodiments, the expression of the gene of interest (e.g., antigen) is increased or elevated by about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold. In some embodiments, the expression of the gene of interest (e.g., antigen) is increased or not increased by at least, up to, just below, or between (inclusive or exclusive) 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, or 50-fold, or any range or value derivable therein.

In some embodiments, the increased expression (e.g., increased duration of expression) of the gene of interest (e.g., antigen) persists at least, up to, just below, or between (including or exclusive of) 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression of the gene of interest (e.g., antigen) continues for at least 24 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) continues for at least 48 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 72 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 96 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) lasts at least 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide.

In some embodiments, the increased expression of the gene of interest (e.g., antigen) is sustained or sustained for about 24-120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increased expression is sustained or sustained about 24-110 hours, 24-100 hours, 24-90 hours, 24-80 hours, 24-70 hours, 24-60 hours, 24-50 hours, 24-40 hours, 24-30 hours, 30-120 hours, 40-120 hours, 50-120 hours, 60-120 hours, 70-120 hours, 80-120 hours, 90-120 hours, 100-120 hours, or 110-120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least, at most, just below, or between (including or exclusive of) 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or any range or value derivable therein.

VII immune response and analysis

As discussed herein, the invention relates to eliciting or inducing an immune response in an individual against an RSV protein (e.g., a wild-type or variant RSV F protein). In one embodiment, the immune response may protect an individual from, or treat an individual having, suspected of having, or at risk of having, an infection or related disease (particularly those associated with RSV). One use of the immunogenic compositions of the invention is to prevent RSV infection by vaccinating or vaccinating an individual. In some embodiments, the immunogenic composition immunizes the individual with RSV for up to 1 year (e.g., for a single RSV season). In some embodiments, the immunogenic composition immunizes the individual for up to 2 years. In some embodiments, the immunogenic composition immunizes the individual with RSV for more than 2 years. In some embodiments, the immunogenic composition immunizes the individual with RSV for more than 3 years. In some embodiments, the immunogenic composition immunizes the individual with RSV for more than 4 years. In some embodiments, the immunogenic composition immunizes the individual with RSV for 5-10 years.

[ A ] immunoassay ]

The invention includes performing a serological analysis to assess whether and to what extent an immune response is induced or induced by the compositions of the invention. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. patent 4,367,110 (double monoclonal antibody sandwich assays) and U.S. patent 4,452,901 (western blot). Other assays include immunoprecipitation and immunocytochemistry of labeled ligands in vitro and in vivo.

Immunoassays are typically binding assays. In some embodiments, immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and Radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one embodiment, the antibody or antigen is immobilized on a selected surface, such as a well in a polystyrene microtiter plate, a dipstick, or a column carrier. Next, a test composition (such as a clinical sample) suspected of containing the desired antigen or antibody is added to the well. After binding and washing to remove non-specifically bound immune complexes, bound antigen or antibody may be detected. Detection is typically accomplished by the addition of another antibody specific for the desired antigen or antibody linked to a detectable label. This type of ELISA is called "sandwich ELISA". Detection may also be achieved by adding a second antibody specific for the desired antigen followed by a third antibody having binding affinity for the second antibody, wherein the third antibody is linked to a detectable label.

Competition ELISA is also a possible embodiment in which the test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeling species prior to or during incubation with the coated wells. The presence of the reactive species in the sample serves to reduce the amount of labeled species available for binding to the well and thus reduce the final signal. Regardless of the format employed, ELISA has certain common features such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes.

The antigen or antibody may also be attached to a solid support, such as in the form of a disc, bead, dipstick, membrane or column matrix, and the sample to be analysed is applied to the immobilised antigen or antibody. When the culture dish is coated with antigen or antibody, the wells of the culture dish are typically incubated with the antigen or antibody solution overnight or for a specified period of time. The wells of the tray are then washed to remove incompletely adsorbed material. Any remaining available surface of the well is then "coated" with a non-specific protein that is antigen neutral to the test antisera. The proteins include Bovine Serum Albumin (BSA), casein and milk powder solutions. This coating allows blocking of non-specific adsorption sites on the immobilized surface and thus reduces the background caused by non-specific binding of antisera on the surface.

[ Diagnosis of RSV infection ]

The present invention encompasses the use of RSV polypeptides, proteins and/or peptides in a variety of ways, including detecting the presence of RSV to diagnose infection. According to the invention, the method of detecting the presence of an infection involves a step of obtaining a sample suspected of being infected with one or more RSV strains, such as a sample obtained from an individual, e.g. a sample obtained from blood, saliva, tissue, bone, muscle, cartilage or skin. After isolating the sample, diagnostic assays utilizing the polypeptides, proteins and/or peptides of the invention can be performed to detect the presence of RSV, and such assay techniques for determining such presence in a sample are well known to those of skill in the art and include methods such as radioimmunoassays, western blot assays, and ELISA assays.

In general, according to the present invention, a method of diagnosing an infection is contemplated, wherein a polypeptide, protein or peptide according to the present invention has been added to a sample suspected of being infected with RSV, and RSV is indicated by binding to an antibody of said polypeptide, protein and/or peptide or binding to an antibody in the sample.

Also encompassed is a method of testing a sample to which a polypeptide, protein or peptide according to the invention has been added, suspected of being infected with RSV, previously infected with RSV or infected with RSV, and RSV is indicated by binding to an antibody to said polypeptide, protein and/or peptide or binding to a polypeptide, protein and/or peptide of an antibody in the sample.

Thus, RNA molecules encoding RSV polypeptides, proteins and/or peptides according to the invention can be used to treat, prevent or reduce the severity of diseases caused by infection by RSV infection (e.g., active or passive immunization) or as a research tool.

Any of the above polypeptides, proteins, and/or peptides may be directly labeled with a detectable label in order to identify and quantify RSV. Labels suitable for immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent, and chromogenic substances, including colored particles, such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

[ C. protective immunity ]

In some embodiments of the invention, RNA molecules encoding RSV preF polypeptides, RNA-LNPs, and compositions thereof confer protective immunity to an individual. Protective immunity refers to the physical ability to establish a specific immune response that protects an individual from a particular disease or condition, which involves an agent against which an immune response exists. An immunogenically effective amount is capable of conferring protective immunity to an individual. In some embodiments, the RNA molecules encoding RSV polypeptides, RNA-LNPs, and compositions thereof of the invention are useful for inducing balanced immune responses (including cellular and humoral immunity) against RSV without many of the risks associated with attenuated viral vaccination. "humoral" immune response refers to an immune response mediated by antibody molecules, including, for example, secretory (IgA) or IgG molecules, while "cellular" immune response is an immune response mediated by T lymphocytes (e.g., CD4 ⁺ helper T cells and/or CD8 ⁺ T cells (e.g., CTLs) and/or other leukocytes.

As used herein, the phrase "immune response" or its equivalent phrase "immune response (immunological response)" refers to the generation of a humoral response (antibody-mediated), a cellular response (mediated by antigen-specific T cells or secreted products thereof), or both a humoral and cellular response to an antigen. Such a reaction may be an active reaction or a passive reaction. Cellular immune responses are initiated by presentation of polypeptide epitopes in combination with class I or class II MHC molecules to activate antigen-specific CD4 (+) T helper cells and/or CD8 (+) cytotoxic T cells. The response may also involve activation of mononuclear spheres, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of the innate immunity. As used herein, "active immunization" refers to any immunization imparted to an individual by the production of antibodies in response to the presence of an antigen (e.g., RSV F protein encoded by an RNA molecule of the invention).

As used herein, "passive immunization" includes, but is not limited to, administration of activated immune effectors, including cellular mediators of immune response or protein mediators (e.g., monoclonal and/or polyclonal antibodies). The single or multiple antibody compositions can be used for passive immunization to treat, prevent, or reduce the severity of a disease caused by infection with an organism carrying the antigen recognized by the antibody. The antibody compositions can include antibodies that bind to a variety of antigens, which can in turn bind to a variety of organisms. The antibody component may be a multi-strain antiserum. In certain embodiments, one or more antibody lines are affinity purified from an animal or a second individual that has been challenged with one or more antigens. Alternatively, a mixture of antibodies may be used, which is a mixture of single and/or multiple antibodies to antigens present in the same, related or different microorganisms or organisms, such as viruses, including but not limited to RSV.

Passive immunization may be conferred on a patient or individual by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from donors or other non-patient sources of known immunoreactivity. In other embodiments, the immunogenic compositions of the invention may be administered to an individual who then acts as a source or donor of globulins containing antibodies to RSV or other organisms that are produced in response to challenge by the immunogenic composition ("hyperimmune"). The individual thus treated will be supplied with plasma from which the hyperimmune immunoglobulin is then obtained via conventional plasma isolation methods and administered to another individual to confer resistance to or treat RSV infection.

For the purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen that B and/or T cells respond to or recognize. B cell epitopes can be formed by contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by consecutive amino acids are usually retained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are usually disappeared after treatment with denaturing solvents. Epitopes typically comprise at least 3 and more typically at least 5 or 8-10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., epitope Mapping Protocols (1996). Antibodies recognizing the same epitope can be identified in a simple immunoassay that shows the ability of one antibody to block the binding of another antibody to the antigen of interest. T cells recognize a contiguous epitope of about nine amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells recognizing an epitope can be identified by in vitro assays that measure antigen dependent proliferation as determined by ³ H-thymidine incorporation by primed T cells in response to the epitope (Burke et al, 1994), by antigen dependent killing (cytotoxic T lymphocyte assay, tigges et al, 1996), or by cytokine secretion.

The presence of a cell-mediated immune response can be determined by proliferation assays (CD 4 (+) T cells) or CTL (cytotoxic T lymphocytes) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effects of an immunogenic composition can be distinguished by isolating IgG and T cells from immunized isotype animals, respectively, and measuring the protective or therapeutic effects in a second individual.

As used herein, the term "antibody" or "immunoglobulin" is used interchangeably and refers to any one of several classes of structurally related proteins, including IgG, igD, igE, igA, igM and related proteins, that function as part of an immune response in an animal or recipient. Under normal physiological conditions, antibodies are present in plasma and other body fluids, as well as in the membranes of certain cells, and are produced by lymphocytes of the type indicated as B cells or functional equivalents thereof.

As used herein, the term "immunogenic agent" or "immunogen" or "antigen" is used interchangeably to describe a molecule capable of inducing an immune response against itself when administered to a recipient alone, in combination with an adjuvant, or presented on a presentation vehicle.

[ VIII ] composition

In some embodiments, the RNA molecules and/or RNA-LNPs disclosed herein can be administered in the form of a pharmaceutical composition or medicament and can be administered in any suitable form of pharmaceutical composition. In some embodiments, the pharmaceutical composition is for therapeutic and/or prophylactic treatment. In one embodiment, the invention relates to a composition for administration to a host. In some embodiments, the host is a human. In other embodiments, the host is a non-human.

Formulations of the vaccine compositions described herein may be prepared by any method known in the pharmacological arts or developed hereafter. Generally, such preparation methods include the steps of combining the active ingredient (e.g., RNA molecule and/or RNA-LNP) with excipients and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, partitioning, shaping and/or packaging the product into the desired single or multi-dose units. The pharmaceutical compositions or formulations according to the present invention may be prepared, packaged and/or sold in bulk form, in single unit dosage form and/or in multiple single unit dosage forms. The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and/or any additional ingredients in the pharmaceutical composition according to the present invention will vary depending upon the identity, size, and/or condition of the individual being treated and further depending upon the route of administration of the composition. By way of example, the composition may comprise between 0.1% and 100%, for example between 0.5% and 50%, between 1% and 30%, between 5% and 80%, at least 80% (w/w), or at least, up to, just below, or between any two of the following (inclusive or exclusive) active ingredient ：0.1％、0.15％、0.2％、0.25％、0.3％、0.35％、0.4％、0.45％、0.5％、0.55％、0.6％、0.65％、0.7％、0.75％、0.8％、0.85％、0.9％、0.95％、1％、5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％ or 100% (w/w). General considerations in the formulation and/or manufacture of pharmaceutical agents such as the compositions described herein can be found, for example, in Remington, THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams and Wilkins,2005 (incorporated herein by reference in its entirety).

In some embodiments, the RNA molecules and/or RNA-LNPs disclosed herein can be administered in the form of a pharmaceutical composition that can be formulated into a solid, semi-solid, liquid, lyophilized, frozen, and/or gaseous form of the formulation. In some embodiments, the RNA molecules and/or RNA-LNPs disclosed herein can be administered in the form of a pharmaceutical composition that can include a pharmaceutically acceptable carrier and optionally one or more adjuvants, stabilizers, salts, buffers, preservatives, and other therapeutic agents, if present. In some embodiments, the pharmaceutical compositions disclosed herein comprise one or more pharmaceutically acceptable carriers, diluents, and/or excipients. In some embodiments, the pharmaceutical composition does not include an adjuvant (e.g., it does not contain an adjuvant).

As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical composition of the present invention but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, diluents (e.g., solvents, dispersion media, and/or other liquid vehicles, dispersions, or suspension aids), granulating and/or dispersing agents, surfactants, isotonic agents, thickening and/or emulsifying agents, preservatives, binders, lubricants and/or oils, colorants, sweeteners and/or flavoring agents, stabilizers, antioxidants, antimicrobial and/or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelating agents, cryoprotectants, and/or bulking agents. In some embodiments, 1,2, 3,4, 5 or more of the foregoing excipients may be excluded from the pharmaceutical compositions disclosed herein.

The term "carrier" refers to a component, which may be natural, synthetic, organic or inorganic, in which the active component is incorporated in order to facilitate, enhance and/or effect administration of the pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to an individual. Suitable carriers include, but are not limited to, sterile water, ringer's solution, ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of the present invention comprise sodium chloride. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing carriers may be excluded from the pharmaceutical compositions disclosed herein.

The term "diluent" refers to a diluting and/or thinning agent. Furthermore, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and/or a mixing medium. Examples of diluents suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, ethanol, glycerol, saline, water, calcium or sodium carbonate, calcium phosphate, dibasic calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, and the like, and/or combinations thereof. In some embodiments, 1, 2,3, 4, 5 or more of the foregoing diluents may be excluded from the pharmaceutical compositions disclosed herein.

Examples of suitable granulating and/or dispersing agents include, but are not limited to, starch, pregelatinized starch, or microcrystalline starch, alginic acid, guar gum, agar, poly (vinyl-pyrrolidone) (povidone)), crosslinked poly (vinyl-pyrrolidone) (crospovidone), cellulose, methylcellulose, carboxymethylcellulose, sodium crosslinked carboxymethylcellulose (croscarmellose), magnesium aluminum silicateSodium lauryl sulfate, and the like, and/or combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing granulating and/or dispersing agents may be excluded from the pharmaceutical compositions disclosed herein.

Examples of surfactants suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, carrageenans, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes and lecithins), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [ s ]80], Sorbitan monopalmitate [40], Glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g.)) Polyoxyethylene ethers (e.g. polyoxyethylene lauryl ether ]30])、F 68、188, Etc., and/or combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing surfactants may be excluded from the pharmaceutical compositions disclosed herein.

Examples of preservatives suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, alkyl dimethylbenzyl ammonium chloride, chlorobutanol, parabens, thimerosal, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxymethoxybenzene, ethylenediamine, sodium Lauryl Sulfate (SLS), sodium Lauryl Ether Sulfate (SLES), and the like, and combinations thereof. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing preservatives may be excluded from the pharmaceutical compositions disclosed herein.

Examples of antimicrobial and/or antifungal agents suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof. In some embodiments, 1,2, 3,4, 5 or more of the foregoing antimicrobial and/or antifungal agents may be excluded from the pharmaceutical compositions disclosed herein.

Examples of binders suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropylmethyl cellulose, and the like, and combinations thereof. In some embodiments, 1, 2, 3, 4,5 or more of the foregoing binders may be excluded from the pharmaceutical compositions disclosed herein.

Examples of lubricants and/or oils suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, and the like, and combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing lubricants and/or oils may be excluded from the pharmaceutical compositions disclosed herein.

Examples of antioxidants suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxymethoxybenzene, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium metabisulfite or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, and the like, and combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing antioxidants may be excluded from the pharmaceutical compositions disclosed herein.

Examples of osmolality adjusting agents, pH adjusting agents, and buffers suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium malate, sodium carbonate, and the like, and/or combinations thereof. In some embodiments, 1, 2, 3,4, 5 or more of the foregoing osmolality adjusting agents may be excluded from the pharmaceutical compositions disclosed herein.

Examples of chelating agents suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, ethylenediamine tetraacetic acid (EDTA), citric acid monohydrate, disodium ethylenediamine tetraacetate, fumaric acid, malic acid, phosphoric acid, sodium ethylenediamine tetraacetate, tartaric acid, trisodium ethylenediamine tetraacetate, and the like, and combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing chelators may be excluded from the pharmaceutical compositions disclosed herein.

Examples of cryoprotectants suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and the like, and combinations thereof. In some embodiments, 1,2, 3, 4, 5 or more of the foregoing cryoprotectants may be excluded from the pharmaceutical compositions disclosed herein.

Examples of suitable bulking agents include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing bulking agents may be excluded from the pharmaceutical compositions disclosed herein.

The compositions may be formulated, for example, using one or more excipients (e.g., one or more carriers and/or diluents) to (1) increase stability, (2) increase cell transfection, (3) allow sustained and/or delayed release (e.g., from a depot formulation), (4) alter biodistribution (e.g., targeting a particular tissue and/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 some embodiments, 1,2, 3, 4, 5, or more of the aforementioned excipient objectives may be excluded. Pharmaceutically acceptable excipients (e.g., carriers and/or diluents) for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, mack Publishing co. (A.R Gennaro editions 1985).

Pharmaceutical excipients (e.g., carriers and/or diluents) may be selected with respect to the intended route of administration and standard pharmaceutical practice.

In some embodiments, the composition comprises an RNA molecule comprising an open reading frame encoding an immunogenic polypeptide. In some embodiments, the immunogenic polypeptide comprises an RSV antigen. In some embodiments, the RSV antigen is an RSV F protein or fragment or variant thereof.

In some embodiments, the composition comprises an RNA molecule comprising an open reading frame encoding a full length RSV F protein. In some embodiments, the encoded immunogenic polypeptide is a truncated RSV F protein. In some embodiments, the encoded immunogenic polypeptide is a variant of RSV F protein. In some embodiments, the encoded immunogenic polypeptide is a fragment of RSV F protein.

[ A ] immunogenic compositions comprising LNP

In some embodiments, the pharmaceutical composition comprises an RNA molecule (e.g., polynucleotide) disclosed herein formulated with a lipid-based delivery system. Thus, in some embodiments, the composition includes a lipid-based delivery system (e.g., LNP) (e.g., lipid-based vaccine) that delivers a nucleic acid molecule to the interior of a cell, which can then replicate, inhibit expression of, and/or express encoded polypeptide of interest within the cell. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition comprises at least one RNA molecule encoding an RSV polypeptide complexed with, encapsulated in, and/or formulated with one or more lipids, and forming a Lipid Nanoparticle (LNP), liposome, lipid complex, and/or nanoliposome. In some embodiments, the composition comprises lipid nanoparticles. Thus, in certain embodiments, the invention relates to compositions comprising one or more lipids (e.g., RSV RNA-LNP) that bind to nucleic acids or polypeptides/peptides.

In some cases, the immunogenic composition comprising the lipid-based delivery system may further comprise one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents and/or excipients. In some embodiments, the immunogenic composition comprising the lipid-based delivery system further comprises a pharmaceutically acceptable vehicle. In some embodiments, each of the buffer, the stabilizer, and optionally the salt may be included in an immunogenic composition comprising a lipid-based delivery system. In other embodiments, any one or more of buffers, stabilizers, salts, surfactants, preservatives, and excipients may be excluded from the immunogenic composition comprising the lipid-based delivery system.

In another embodiment, the immunogenic composition comprising a lipid-based delivery system further comprises a stabilizer. In some embodiments, the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, polydextrose, polyvinylpyrrolidone, glycine, or a combination thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing stabilizers may be excluded from the immunogenic compositions disclosed herein. In some embodiments, the stabilizer is a disaccharide or sugar. In one embodiment, the stabilizer is sucrose. In another embodiment, the stabilizing agent is trehalose. In another embodiment, the stabilizing agent is a combination of sucrose and trehalose. In some embodiments, the total concentration of the one or more stabilizers in the composition is at or about 5% to about 10% w/v. For example, the total concentration of the stabilizers may or may not be at least, up to, exactly equal to, or between (inclusive or exclusive) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v, or any range or value derivable therein. In some embodiments, the stabilizer concentration includes, but is not limited to, a concentration of or from about 10mg/mL to about 400mg/mL, about 100mg/mL to about 200mg/mL, about 100mg/mL to about 150mg/mL, about 100mg/mL to about 140mg/mL, about 100mg/mL to about 130mg/mL, about 100mg/mL to about 120mg/mL, about 100mg/mL to about 110mg/mL, or about 100mg/mL to about 105 mg/mL. In some embodiments, the concentration of the stabilizer is or is not at least, at most, exactly equal to or between (inclusive or exclusive )：10mg/mL、20mg/mL、50mg/mL、100mg/mL、101mg/mL、102mg/mL、103mg/mL、104mg/mL、105mg/mL、106mg/mL、107mg/mL、108mg/mL、109mg/mL、110mg/mL、150mg/mL、200mg/mL、300mg/mL、400mg/mL or higher).

In another embodiment, the amount of the mass of the stabilizing agent is in a specific ratio to the amount of the mass of the RNA. In one embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 5000. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 2000. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 1000. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 500. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 100. In another embodiment, the ratio of the amount of stabilizer to the mass of the pharmaceutical substance does not exceed 50. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 10. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 1. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 0.5. In another embodiment, the ratio of the amount of stabilizer to the mass of RNA does not exceed 0.1. In another embodiment, the stabilizer and RNA comprise a mass ratio of stabilizer to RNA of about 200 to 2000:1.

In some embodiments, the immunogenic composition comprising the lipid-based delivery system further comprises a buffer. Examples of buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium glucoheptonate, calcium gluconate, d-gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, calcium phosphate hydroxide, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, ammonium bradykinin, tris hydrochloric acid (HCl), sulfamate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, etc Zhang Yanshui, ringer's solution, ethanol, and/or combinations thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing buffers may be excluded from the immunogenic compositions disclosed herein. In some embodiments, the buffer is HEPES buffer, tris buffer, and/or PBS buffer. In one embodiment, the buffer is Tris buffer. In another embodiment, the buffer is HEPES buffer. In another embodiment, the buffer is a PBS buffer. For example, the buffer concentration may or may not be at least, up to, exactly equal to, or between (inclusive or exclusive) 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, or 20mM, or any range or value derivable therein. The buffer solution can be neutral pH, pH 6.5-8.5, pH 7.0-8.0, or pH 7.2-7.6. For example, the buffer may or may not be at least, up to, just below or between (inclusive or exclusive) pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In a particular embodiment, the buffer is at pH 7.4.

In some embodiments, the immunogenic composition comprising the lipid-based delivery system may further comprise a salt. Examples of salts include, but are not limited to, sodium and/or potassium salts. In one embodiment, the salt is a sodium salt. In a particular embodiment, the sodium salt is sodium chloride. In one embodiment, the salt is a potassium salt. In some embodiments, the potassium salt comprises potassium chloride. In some embodiments, any one or more of the foregoing salts may be excluded from the immunogenic compositions disclosed herein. The concentration of salt in the composition may be at or about 70mM to about 140mM. For example, the salt concentration may or may not be at least, up to, exactly equal to, or between (inclusive or exclusive) 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM.

In some embodiments, the salt concentration includes, but is not limited to, a concentration of or from about 1mg/mL to about 100mg/mL, about 1mg/mL to about 50mg/mL, about 1mg/mL to about 40mg/mL, about 1mg/mL to about 30mg/mL, about 1mg/mL to about 20mg/mL, about 1mg/mL to about 10mg/mL, or about 1mg/mL to about 15 mg/mL. In some embodiments, the concentration of salt is or is not at least, at most, exactly equal to or between (including or exclusive )：1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL、6mg/mL、7mg/mL、8mg/mL、9mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL or higher. Salt may be neutral pH, pH 6.5-8.5, pH 7.0-pH 8.0, or pH 7.2-pH 7.6. For example, salt may or may not be at least, at most, exactly equal to or between (including or exclusive) pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.

In some embodiments, the immunogenic composition comprising the lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof. As used herein, "any other excipient" includes, but is not limited to, antioxidants, glutathione, EDTA, methionine, deferoxamine mesylate (desferal), antioxidants, metal scavengers, and/or radical scavengers. In one embodiment, the surfactant, preservative, excipient, or combination thereof is sterile water for injection (sWFI), bacteriostatic water for injection (BWFI), saline, dextrose solution, polysorbate, poloxamer (poloxamer), triamcinolone, divalent cations, ringer's lactate, amino acids, sugars, polyols, polymers, and/or cyclodextrins. In some embodiments, 1,2,3, 4, 5 or more of the foregoing excipients may be excluded from the immunogenic compositions disclosed herein.

Excipients refer to ingredients of the immunogenic composition that are not active ingredients, other examples of which include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compressing agents, wet granulating agents, and/or coloring agents. As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles such as sodium chloride, ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, adhesives, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, fluids, and nutritional supplements such as will be known to those of ordinary skill in the art, and combinations thereof. Diluents or diluents include, but are not limited to, ethanol, glycerol, water, sugars (such as lactose, sucrose, mannitol and sorbitol) and starches derived from wheat, corn rice and potato, and celluloses such as microcrystalline cellulose. The amount of diluent in the composition may be in the range of or about 10% to about 90%, for example in the range of or about 25% to about 75%, about 30% to about 60%, or about 12% to about 60% by weight of the total composition. Preservatives for use in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. In some embodiments, 1,2, 3,4, 5 or more of the foregoing excipients may be excluded from the immunogenic compositions disclosed herein.

The pH and exact concentration of the various components in the immunogenic composition, including the lipid-based delivery system, are adjusted according to well known parameters. The use of such media and medicaments for pharmaceutically active substances is well known in the art. Unless any conventional medium or agent is incompatible with the active ingredient, its use in immunogenic, prophylactic and/or therapeutic compositions is contemplated.

In one embodiment, the pharmaceutical composition comprises an RSV RNA molecule encoding an RSV polypeptide disclosed herein that is complexed with, encapsulated in, and/or formulated with one or more lipids to form an RSV RNA-LNP. In some embodiments, the RSV RNA-LNP composition is a liquid. In some embodiments, the RSV RNA-LNP composition is frozen. In some embodiments, the RSV RNA-LNP composition is lyophilized. In some embodiments, the RSV RNA-LNP compositions comprise an RSV RNA polynucleotide molecule encoding an RSV polypeptide disclosed herein encapsulated in an LNP having a lipid composition of a cationic lipid, a pegylated lipid (i.e., a PEG-lipid), and one or more structural lipids (e.g., a neutral lipid). In some embodiments, any one or more of the foregoing lipids may be excluded from the LNP of the pharmaceutical compositions disclosed herein.

In some embodiments, the RSV RNA-LNP composition comprises a cationic lipid. The cationic lipid may comprise any one or more of the cationic lipids disclosed herein.

The cationic lipid may comprise any one or more of the cationic lipids disclosed herein. In a particular embodiment, the cationic lipid comprises ((4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (ALC-0315). In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or without at least, at most, between (including or exclusive of) or just below a concentration of ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99 or 2ng/μg/mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or without at least, up to, between (inclusive or exclusive) or just below a concentration of ：0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99 or 1mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least 0.4mg/mL, at least 0.45mg/mL, at least 0.5mg/mL, at least 0.55mg/mL, at least 0.6mg/mL, at least 0.65mg/mL, at least 0.7mg/mL, at least 0.75mg/mL, at least 0.8mg/mL, at least 0.85mg/mL, at least 0.9mg/mL, at least 0.95mg/mL, or at least 1mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.5mg/mL, between 0.5 and 0.6mg/mL, between 0.6 and 0.7mg/mL, between 0.7 and 0.8mg/mL, between 0.8 and 0.9mg/mL, or between 0.9 and 1mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.45mg/mL, between 0.45 and 0.5mg/mL, between 0.5 and 0.55mg/mL, between 0.55 and 0.6mg/mL, between 0.6 and 0.65mg/mL, between 0.65 and 0.7mg/mL, between 0.7 and 0.75mg/mL, between 0.75 and 0.8mg/mL, between 0.8 and 0.85mg/mL, between 0.85 and 0.9mg/mL, between 0.9 and 0.95mg/mL, or between 0.95 and 1mg/mL.

In certain embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8-0.95 mg/mL. In particular embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or in a concentration of about 0.8-0.9 mg/mL. In particular embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or in a concentration of about 0.85-0.9 mg/mL. In particular embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or without a concentration below or at least, up to, just below or between (including or exclusive of) or about below any two of 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94 or 0.95mg/mL. In particular embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at or in a concentration of about 0.86 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some embodiments, the RSV RNA-LNP composition further comprises a pegylated lipid (i.e., PEG-lipid).

The pegylated lipid may comprise any one or more of the pegylated lipids disclosed herein. In a particular embodiment, the pegylated lipid comprises 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159). In some embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or without at least below, up to below, between (including or exclusive of) or just below, ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99 or 2ng/μg/mg/mL. In some embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or without at least below, up to below, between (including or exclusive of) or just below, ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49 or 0.5mg/mL. In some embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least 0.01mg/mL, at least 0.05mg/mL, at least 0.1mg/mL, at least 0.15mg/mL, at least 0.2mg/mL, at least 0.25mg/mL, at least 0.3mg/mL, at least 0.35mg/mL, at least 0.4mg/mL, at least 0.45mg/mL, or at least 0.5mg/mL. In some embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of between 0.01 and 0.05mg/mL, between 0.05 and 0.1mg/mL, between 0.1 and 0.15mg/mL, between 0.15 and 0.2mg/mL, or between 0.2 and 0.25 mg/mL.

In particular embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or in a concentration of about 0.05 to 0.15mg/mL. In particular embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or in a concentration of about 0.10 to 0.15mg/mL. In particular embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or without a concentration of 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15mg/mL, or at least, up to, just below, or between (including or exclusive of) any two of the following. In particular embodiments, the pegylated lipid (e.g., ALC-0159) is included in the composition at or at a concentration of about 0.11 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some embodiments, the RSV RNA-LNP composition further comprises one or more structural lipids.

The one or more structural lipids may comprise any one or more of the structural lipids disclosed herein. In particular embodiments, the one or more structural lipids comprise neutral lipids and steroids or steroid analogues. In particular embodiments, the one or more structural lipids comprise 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and cholesterol. In some embodiments, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at or without at least below, at most below, between (inclusive or exclusive) below, or just below, at a concentration of ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99 or 2ng/μg/mg/mL. In some embodiments, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at or without at least below, at most below, between (inclusive or exclusive) below, or just below, at a concentration of ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49 or 0.5mg/mL. In some embodiments, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least 0.05mg/mL, at least 0.1mg/mL, at least 0.15mg/mL, at least 0.2mg/mL, at least 0.25mg/mL, at least 0.3mg/mL, at least 0.35mg/mL, at least 0.4mg/mL, at least 0.45mg/mL, at least 0.5mg/mL, at least 0.55mg/mL, at least 0.6mg/mL, at least 0.65mg/mL, at least 0.7mg/mL, at least 0.75mg/mL, at least 0.8mg/mL, at least 0.85mg/mL, at least 0.9mg/mL, at least 0.95mg/mL, or at least 1 mg/mL. In some embodiments, the one or more structural lipids (e.g., DSPC and cholesterol) are included in a concentration of between 0.05 and 0.1mg/mL, between 0.1 and 0.15mg/mL, between 0.15 and 0.2mg/mL, between 0.2 and 0.25mg/mL, between 0.25 and 0.3mg/mL, between 0.3 and 0.35mg/mL, between 0.35 and 0.4mg/mL, between 0.4 and 0.45mg/mL, between 0.45 and 0.5mg/mL, between 0.5 and 0.55mg/mL, between 0.55 and 0.6mg/mL, between 0.6 and 0.65mg/mL, between 0.65 and 0.7mg/mL, between 0.7 and 0.75mg/mL, between 0.75 and 0.8mg/mL, between 0.8 and 0.85mg/mL, between 0.85 and 0.9mg/mL, between 0.9 and 0.9mg/mL, between 0.95mg/mL, or between 1.95 mg/mL.

In particular embodiments, the one or more structural lipids comprise DSPC, and the DSPC is included in the composition at or in a concentration of about 0.1-0.25 mg/mL. In particular embodiments, the one or more structural lipids comprise DSPC, and the DSPC is included in the composition at or in a concentration of about 0.15-0.25 mg/mL. In particular embodiments, the one or more structural lipids comprise DSPC and DSPC is included in the composition in a concentration of 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, or 0.25mg/mL, or at least, up to, just below, or between (including or exclusive of) any two of the following. In particular embodiments, the DSPC is included in the composition at or in a concentration of about 0.19 mg/mL.

In particular embodiments, the one or more structural lipids comprise cholesterol, and cholesterol is included in the composition at or in a concentration of about 0.3-0.45 mg/mL. In particular embodiments, the one or more structural lipids include cholesterol, and cholesterol is included in the composition at or in a concentration of about 0.3-0.4 mg/mL. In particular embodiments, the one or more structural lipids comprise cholesterol, and cholesterol is included in the composition at or at a concentration of about 0.35-0.45 mg/mL. In particular embodiments, the one or more structural lipids include cholesterol and cholesterol is included in the composition at or without a concentration below or at least, up to, just below or between (including or exclusive of) or about below any two of 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44 or 0.45mg/mL. In particular embodiments, cholesterol is included in the composition at and/or at a concentration of about 0.37 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some embodiments, the RSV RNA-LNP composition further comprises one or more buffering agents and stabilizers and, optionally, a salt diluent. Thus, in some embodiments, the RSV RNA-LNP composition comprises a cationic lipid, a pegylated lipid, one or more structural lipids, one or more buffers, a stabilizer, and optionally a salt diluent. In some embodiments, 1, 2, 3, or more of the foregoing elements are excluded from the RSV RNA-LNP composition.

In some embodiments, the RSV RNA-LNP composition comprises one or more buffers.

The one or more buffers may comprise any one or more of the buffers disclosed herein. In a particular embodiment, the composition comprises Tris buffer comprising at least a first buffer and a second buffer. In some embodiments, the first buffer is tromethamine. In some embodiments, the second buffer is Tris hydrochloric acid (HCl). In some embodiments, the first and second buffers of the Tris buffer (e.g., bradykinin and Tris HCl) are included in the composition or are not included in a concentration of at least below, at most below, between (inclusive or exclusive) below, or just below, ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99 or 2ng/μg/mg/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some embodiments, the RSV RNA-LNP composition is a liquid composition comprising Tris buffer.

In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is tromethamine. In some embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at or without at least below, up to below, between (inclusive or exclusive) below, or just below, ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49 or 0.5mg/mL. In some embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of at least 0.1mg/mL, at least 0.05mg/mL, at least 0.1mg/mL, at least 0.15mg/mL, at least 0.2mg/mL, at least 0.25mg/mL, at least 0.3mg/mL, at least 0.35mg/mL, at least 0.4mg/mL, at least 0.45mg/mL, at least 0.5mg/mL, at least 0.55mg/mL, at least 0.6mg/mL, at least 0.65mg/mL, at least 0.7mg/mL, at least 0.75mg/mL, at least 0.8mg/mL, at least 0.85mg/mL, at least 0.9mg/mL, at least 0.95mg/mL, or at least 1 mg/mL. In some embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of between 0.05 and 0.15mg/mL, between 0.15 and 0.25mg/mL, between 0.25 and 0.35mg/mL, between 0.35 and 0.45mg/mL, between 0.45 and 0.55mg/mL, between 0.55 and 0.65mg/mL, between 0.65 and 0.75mg/mL, between 0.75 and 0.85mg/mL, or between 0.85 and 0.95 mg/mL. In some embodiments, the first buffer (e.g., tromethamine) comprises a liquid at a concentration between 0.05 and 0.1mg/mL, between 0.1 and 0.15mg/mL, between 0.15 and 0.2mg/mL, between 0.2 and 0.25mg/mL, between 0.25 and 0.3mg/mL, between 0.3 and 0.35mg/mL, between 0.35 and 0.4mg/mL, between 0.4 and 0.45mg/mL, between 0.45 and 0.5mg/mL, between 0.5 and 0.55mg/mL, between 0.55 and 0.6mg/mL, between 0.6 and 0.65mg/mL, between 0.65 and 0.7mg/mL, between 0.7 and 0.75mg/mL, between 0.75 and 0.8mg/mL, between 0.8 and 0.85mg/mL, between 0.85 and 0.9mg/mL, between 0.9 and 0.95mg/mL, between 0.95mg/mL, or between 1 and 0.95 mg/mL.

In certain embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at or at a concentration of about 0.1-0.3 mg/mL. In certain embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at or at a concentration of about 0.15-0.25 mg/mL. In particular embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at or without a concentration of 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3mg/mL, or at least, up to, just below, or between (including or exclusive of) any two of the following. In particular embodiments, the first buffer (e.g., tromethamine) is included in the liquid composition at or at a concentration of about 0.20 mg/mL.

In some embodiments, the RSV RNA-LNP composition is a liquid composition comprising a Tris buffer comprising a second buffer.

In some embodiments, the second buffer comprises Tris HCl. In some embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at or without at least below, up to below, between (inclusive or exclusive) below, or just below, ：0.5、0.55、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49 or 1.5mg/mL. In some embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at a concentration of at least 0.5mg/mL, at least 0.55mg/mL, at least 0.6mg/mL, at least 0.65mg/mL, at least 0.7mg/mL, at least 0.75mg/mL, at least 0.8mg/mL, at least 0.85mg/mL, at least 0.9mg/mL, at least 0.95mg/mL, at least 1mg/mL, at least 1.05mg/mL, at least 1.10mg/mL, at least 1.15mg/mL, at least 1.20mg/mL, at least 1.25mg/mL, at least 1.30mg/mL, at least 1.35mg/mL, at least 1.40mg/mL, at least 1.45mg/mL, or at least 1.50 mg/mL. In some embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at a concentration between 0.5 and 0.6mg/mL, between 0.6 and 0.7mg/mL, between 0.7 and 0.8mg/mL, between 0.8 and 0.9mg/mL, between 0.9 and 1mg/mL, between 1 and 1.10mg/mL, between 1.10 and 1.20mg/mL, between 1.20 and 1.30mg/mL, between 1.30 and 1.40mg/mL, or between 1.40 and 1.50 mg/mL.

In certain embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at or at a concentration of about 1.25-1.40 mg/mL. In certain embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at or at a concentration of about 1.30-1.40 mg/mL. In particular embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at or without a concentration of 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, or 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40mg/mL, or at least, up to, just below, or between any two of the following (inclusive or exclusive) or about below. In particular embodiments, the second buffer (e.g., tris HCl) is included in the liquid composition at or at a concentration of about 1.32 mg/mL.

In some embodiments, the RSV RNA-LNP composition is a lyophilized composition comprising Tris buffer. In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is tromethamine. In some embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or without at least below, up to below, between (including or exclusive of) or just below, at a post reconstitution concentration of ：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49 or 0.5mg/mL. In some embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a post reconstitution concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, or at least 0.5mg/mL. In some embodiments, the first buffer (e.g., bradykinin (Tris base)) is included in the lyophilized composition at a post reconstitution concentration between 0.01 and 0.05mg/mL, between 0.05 and 0.1mg/mL, between 0.1 and 0.15mg/mL, between 0.15 and 0.2mg/mL, between 0.2 and 0.25mg/mL, between 0.25 and 0.3mg/mL, between 0.3 and 0.35mg/mL, between 0.35 and 0.4mg/mL, between 0.4 and 0.45mg/mL, or between 0.45 and 0.5mg/mL.

In particular embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or in a post reconstitution concentration of about 0.01 and 0.15mg/mL. In particular embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or in a post reconstitution concentration of about 0.01 and 0.10 mg/mL. In particular embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or in a post reconstitution concentration of about 0.05 and 0.15mg/mL. In particular embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or without a post-reconstitution concentration of or at least, up to, just below, or between (including or exclusive of) or about below any two of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15mg/mL. In particular embodiments, the first buffer (e.g., tromethamine) is included in the lyophilized composition at or at a post reconstitution concentration of about 0.09 mg/mL.

In some embodiments, the RSV RNA-LNP composition is a lyophilized composition comprising a Tris buffer comprising a second buffer. In some embodiments, the second buffer comprises Tris HCl. In some embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at or without at least, up to, between (including or exclusive of) or just below post reconstitution concentrations of ：0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99 or 1mg/mL. In some embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at a post-reconstitution concentration of at least 0.1mg/mL, at least 0.2mg/mL, at least 0.3mg/mL, at least 0.4mg/mL, at least 0.5mg/mL, at least 0.6mg/mL, at least 0.7mg/mL, at least 0.8mg/mL, at least 0.9mg/mL, or at least 1mg/mL. In some embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at a post-reconstitution concentration of between 0.1 and 0.2mg/mL, between 0.2 and 0.3mg/mL, between 0.3 and 0.4mg/mL, between 0.4 and 0.5mg/mL, between 0.5 and 0.6mg/mL, between 0.6 and 0.7mg/mL, between 0.7 and 0.8mg/mL, between 0.8 and 0.9mg/mL, or between 0.9 and 1mg/mL.

In particular embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at or in a post-reconstitution concentration of about 0.5 and 0.65mg/mL. In particular embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at or in a post-reconstitution concentration of about 0.5 and 0.6 mg/mL. In particular embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at a post-reconstitution concentration of about 0.55 and 0.65mg/mL. In particular embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at or without a post-reconstitution concentration of or at least, up to, just below, or between (including or exclusive of) or about below any two of 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, or 0.65mg/mL. In particular embodiments, the second buffer (e.g., tris HCl) is included in the lyophilized composition at or at a post-reconstitution concentration of about 0.57 mg/mL.

In some embodiments, the RSV RNA-LNP composition comprises a stabilizer. The stabilizer may comprise any one or more of the stabilizers disclosed herein. In some embodiments, the stabilizer also acts as a cryoprotectant. In a particular embodiment, the stabilizing agent comprises sucrose. In some embodiments, a stabilizer (e.g., sucrose) is included in the composition at or without at least, up to, between (inclusive or exclusive) or just below a concentration of ：1、2、3、4、5、6、7、8、9、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、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、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199 or 200ng/μg/mg/mL.

In some embodiments, the RSV RNA-LNP composition is a liquid composition and the stabilizer (e.g., sucrose) is included in the liquid composition at or without at least below, at most below, between (inclusive or exclusive) below, or just below, ：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、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129 or 130mg/mL. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, or at least 130mg/mL. In some embodiments, a stabilizer (e.g., sucrose) is included in the liquid composition at a concentration between 70 and 80mg/mL, between 80 and 90mg/mL, between 90 and 100mg/mL, between 100 and 110mg/mL, between 110 and 120mg/mL, or between 120 and 130mg/mL.

In certain embodiments, a stabilizer (e.g., sucrose) is included in the liquid composition at or at a concentration of about 95-110 mg/mL. In particular embodiments, a stabilizer (e.g., sucrose) is included in the liquid composition at or at a concentration of about 95-105 mg/mL. In certain embodiments, a stabilizer (e.g., sucrose) is included in the liquid composition at or at a concentration of about 100 to 110mg/mL. In particular embodiments, the stabilizing agent (e.g., sucrose) is included in the liquid composition at or without a concentration below or at least, up to, just below, between (including or exclusive of) any two of the following, or about below, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110mg/mL. In particular embodiments, a stabilizer (e.g., sucrose) is included in the liquid composition at or in a concentration of about 103 mg/mL.

In some embodiments, the RSV RNA-LNP composition is a lyophilized composition and the stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or without at least below, at most below, between (including or exclusive of) or just below post reconstitution concentrations of ：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、70、71、72、73、74、75、76、77、78、79 or 80mg/mL. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the lyophilized composition at a post reconstitution concentration of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80mg/mL. In some embodiments, a stabilizing agent (e.g., sucrose) is included in the lyophilized composition at a post reconstitution concentration of between 20-30 mg/mL, between 30-40 mg/mL, between 40-50 mg/mL, between 50-60 mg/mL, between 60-70 mg/mL, or between 70-80 mg/mL.

In particular embodiments, a stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or at a post reconstitution concentration of about 35 to 50mg/mL. In particular embodiments, a stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or at a post reconstitution concentration of about 35 to 45 mg/mL. In particular embodiments, a stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or in a post reconstitution concentration of about 40 to 50mg/mL. In particular embodiments, the stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or without a post-reconstitution concentration or at least, up to, just below, between (including or exclusive of) or about a post-reconstitution concentration of any two of 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50mg/mL. In particular embodiments, a stabilizing agent (e.g., sucrose) is included in the lyophilized composition at or in a post reconstitution concentration of about 44 mg/mL.

In some embodiments, the lyophilized composition is reconstituted in a suitable carrier and/or diluent. The carrier and/or diluent may comprise any one or more of the carriers and/or diluents disclosed herein. In particular embodiments, the carrier and/or diluent comprises a salt diluent, such as sodium chloride (NaCl) (e.g., saline, e.g., physiological saline (physiological/normal saline)). The sodium chloride may comprise 0.9% sodium chloride for injection. In some embodiments, the lyophilized composition is reconstituted ：0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.50、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.60、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.70、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99 or 1mL in saline, or not at least below, up to below, between (inclusive or exclusive) below, or just below. In some embodiments, the lyophilized composition is reconstituted in at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1mL of sodium chloride.

In particular embodiments, the lyophilized composition is reconstituted in or in about 0.6 to 0.75ml sodium chloride/saline. In particular embodiments, the lyophilized composition is reconstituted in or in about 0.65 to 0.75ml sodium chloride/saline. In particular embodiments, the lyophilized composition is reconstituted in sodium chloride/saline, either at least, up to, just below, between any two of (inclusive or exclusive), or about below, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0,74, or 0.75mL.

In some embodiments, a salt diluent (e.g., naCl) is included in the lyophilized composition at or without at least, up to, between (including or exclusive of) or just below, a post reconstitution concentration of 0.5、1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5、11、11.5、12、12.5、13、13.5、14、14.5、15、15.5、16、16.5、17、17.5、18、18.5、19、19.5、20、20.5、21、21.5、22、22.5、23、23.5、24、24.5、25、25.5、26、26.5、27、27.5、28、28.5、29、29.5、30、30.5、31、31.5、32、32.5、33、33.5、34、34.5、35、35.5、36、36.5、37、37.5、38、38.5、39、39.5、40、40.5、41、41.5、42、42.5、43、43.5、44、44.5、45、45.5、46、46.5、47、47.5、48、48.5、49、49.5 or 50 ng/. Mu.g/mg per milliliter. In some embodiments, a salt diluent (e.g., naCl) is included in the lyophilized composition at or without at least, up to, between (including or exclusive of) or just below post reconstitution concentrations of ：1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5、11、11.5、12、12.5、13、13.5、14、14.5、15、15.5、16、16.5、17、17.5、18、18.5、19、19.5 or 20mg/mL. In some embodiments, the salt diluent (e.g., naCl) is included in the lyophilized composition at a post-reconstitution concentration of 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, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20mg/mL.

In particular embodiments, a salt diluent (e.g., naCl) is included in the lyophilized composition at a post-reconstitution concentration of between 5 and 15mg/mL or between about. In some embodiments, a salt diluent (e.g., naCl) is included in the lyophilized composition at a post reconstitution concentration of between 5 and 10mg/mL or between about therebetween. In particular embodiments, the salt diluent (e.g., naCl) is included in the lyophilized composition at or without a post-reconstitution concentration of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15mg/mL or at least, up to, just below, between (including or exclusive of) or about below any two of the following. In particular embodiments, a salt diluent (e.g., naCl) is included in the lyophilized composition at or in a post-reconstitution concentration of about 9 mg/mL.

The pH of the RSV RNA-LNP composition may or may not be at least, up to, just below, or between (inclusive or exclusive) pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In some embodiments, the RSV RNA-LNP composition is at a pH of at least 6.5, at least 7.0, at least 7.5, at least 8.0, or at least 8.5. In particular embodiments, the RSV RNA-LNP composition is at a pH between 6.0 and 7.5, between 6.5 and 7.5, between 7.0 and 8.0, between 7.5 and 8.5. In a particular embodiment, the RSV RNA-LNP composition is at a pH between 7.0 and 8.0. In particular embodiments, the RSV RNA-LNP composition is or is not at least, up to, just below, between (including or exclusive of) or about below pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0. In particular embodiments, the RSV RNA-LNP composition is at or about pH 7.4. In some embodiments, sodium hydroxide buffer may be used for buffer pH adjustment.

In particular embodiments, the RSV RNA-LNP compositions comprise RSV RNA polynucleotides encoding RSV polypeptides disclosed herein encapsulated in LNPs having a lipid composition of cationic lipids at or about 0.8-0.95 mg/mL, pegylated lipids at or about 0.05-0.15 mg/mL, first structural lipids at or about 0.1-0.25 mg/mL, and second structural lipids at or about 0.3-0.45 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In particular embodiments, the RSV RNA-LNP compositions comprise RSV RNA polynucleotides encoding RSV polypeptides disclosed herein encapsulated in LNPs having a lipid composition of ALC-0315 at or about 0.8-0.95 mg/mL, ALC-0159 at or about 0.05-0.15 mg/mL, DSPC at or about 0.1-0.25 mg/mL, and cholesterol at or about 0.3-0.45 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In a particular embodiment, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition, and the liquid RSV RNA-LNP composition further comprises a buffer composition comprising a first buffer at a concentration of or about 0.15 to 0.3mg/mL, a second buffer at a concentration of or about 1.25 to 1.4mg/mL, and a stabilizing agent at a concentration of or about 95 to 110 mg/mL. In particular embodiments, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition, and the liquid RSV RNA-LNP composition further comprises a Tris buffer composition comprising, at or about 0.1 to 0.3mg/mL, tris HCl at or about 1.25 to 1.4mg/mL, and sucrose at or about 95 to 110 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in certain embodiments, the liquid RSVRNA-LNP composition comprises a cationic lipid at or about 0.8-0.95 mg/mL, a pegylated lipid at or about 0.05-0.15 mg/mL, a first structural lipid at or about 0.1-0.25 mg/mL, a second structural lipid at or about 0.3-0.45 mg/mL, and further comprises a first buffer at or about 0.1-0.3 mg/mL, a second buffer at or about 1.25-1.4 mg/mL, and a stabilizer at or about 95-110 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in certain embodiments, the liquid RSVRNA-LNP composition comprises ALC-0315 at a concentration of or about 0.8-0.95 mg/mL, ALC-0159 at a concentration of or about 0.05-0.15 mg/mL, DSPC at a concentration of or about 0.1-0.25 mg/mL, cholesterol at a concentration of or about 0.3-0.45 mg/mL, and further comprises a Tris buffer composition comprising bradykinin at a concentration of or about 0.1-0.3 mg/mL, tris HCl at a concentration of or about 1.25-1.4 mg/mL, and sucrose at a concentration of or about 95-110 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In particular embodiments, the RSV RNA-LNP composition is a lyophilized RSV RNA-LNP composition, and the lyophilized RSV RNA-LNP composition further comprises (after reconstitution) a first buffer at a concentration of or about 0.01 to 0.15mg/mL, a second buffer at a concentration of or about 0.5 to 0.65mg/mL, a stabilizing agent at a concentration of or about 35 to 50mg/mL, and a salt diluent at a concentration of or about 5 to 15mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In particular embodiments, the RSV RNA-LNP composition is a lyophilized RSV RNA-LNP composition, and the lyophilized RSV RNA-LNP composition further comprises (after reconstitution) a Tris buffer composition comprising, at a concentration of or about 0.01 to 0.15mg/mL, tris HCl at a concentration of or about 0.5 to 0.65mg/mL, sucrose at a concentration of or about 35 to 50mg/mL, and sodium chloride (NaCl) at a concentration of or about 5 to 15mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in certain embodiments, the lyophilized RSVRNA-LNP composition comprises (after reconstitution) a cationic lipid at or about 0.8-0.95 mg/mL, a pegylated lipid at or about 0.05-0.15 mg/mL, a first structural lipid at or about 0.1-0.25 mg/mL, a second structural lipid at or about 0.3-0.45 mg/mL, and further comprises a first buffer at or about 0.01-0.15 mg/mL, a second buffer at or about 0.5-0.65 mg/mL, a stabilizer at or about 35-50 mg/mL, and a salt diluent at or about 5-15 mg/mL. In a particular embodiment, the lyophilized composition is reconstituted in 0.6 to 0.75ml of salt diluent. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in some embodiments, the lyophilized RSVRNA-LNP composition comprises (after reconstitution) ALC-0315 at or about 0.8-0.95 mg/mL, ALC-0159 at or about 0.05-0.15 mg/mL, DSPC at or about 0.1-0.25 mg/mL, cholesterol at or about 0.3-0.45 mg/mL, and further comprises bradykinin at or about 0.01-0.15 mg/mL, tris HCl at or about 0.5-0.65 mg/mL, sucrose at or about 35-50 mg/mL, and NaCl at or about 5-15 mg/mL. In a particular embodiment, the lyophilized composition is reconstituted in 0.6 to 0.75ml of NaCl (saline). In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

The concentration in the above lyophilized RSV RNA-LNP compositions was determined after reconstitution.

In some embodiments, the RSV RNA-LNP composition (prior to lyophilization) comprises a cationic lipid at or about 1.0 to 3.0mg/mL, a pegylated lipid at or about 0.10 to 0.35mg/mL, a first structural lipid at or about 0.4 to 0.55mg/mL, a second structural lipid at or about 0.85 to 1.0mg/mL, and further comprises a first buffer at or about 0.1 to 0.3mg/mL, a second buffer at or about 1.25 to 1.40mg/mL, and a stabilizer at or about 95 to 110mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in some embodiments, the RSV RNA-LNP composition (prior to lyophilization) comprises ALC-0315 at a concentration of or about 1.0-3.0 mg/mL, ALC-0159 at a concentration of or about 0.10-0.35 mg/mL, DSPC at a concentration of or about 0.4-0.55 mg/mL, cholesterol at a concentration of or about 0.85-1.0 mg/mL, and further comprises bradykinin at a concentration of or about 0.1-0.3 mg/mL, tris HCl at a concentration of or about 1.25-1.40 mg/mL, sucrose at a concentration of or about 95-110 mg/m. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

The RSV RNA-LNP composition further comprises an RSV RNA described herein encapsulated in an LNP.

In particular embodiments, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition comprising an RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01 to 0.09mg/mL, encapsulated in an LNP having a lipid composition comprising a cationic lipid at or about 0.8 to 0.95mg/mL, a pegylated lipid at or about 0.05 to 0.15mg/mL, a first structural lipid at or about 0.1 to 0.25mg/mL, and a second structural lipid at or about 0.3 to 0.45mg/mL, and further comprising a buffer composition comprising a first buffer at or about 0.15mg/mL, a second buffer at or about 1 to about 4mg/mL, and a stabilizing agent at or about 1 to about 0.95 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In particular embodiments, the liquid RSV RNA-LNP compositions comprise the RSV RNA polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01 to 0.09mg/mL, and more preferably at or about 0.06mg/mL, encapsulated in LNP having a lipid composition of ALC-0315 at or about 0.8 to 0.95mg/mL, ALC-0159 at or about 0.05 to 0.15mg/mL, DSPC at or about 0.1 to 0.25mg/mL, and cholesterol at or about 0.3 to 0.45mg/mL, and further comprising Tris buffer compositions comprising Tris buffer at or about 1.4mg/mL, sucrose at or about 1 to about 1.95 mg/mL, and sucrose at or about 1 to about 4.25 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In particular embodiments, the RSV RNA-LNP composition is a lyophilized RSV RNA-LNP composition comprising an RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01 to 0.09mg/mL, encapsulated in LNP having a lipid composition of a cationic lipid at or about 0.8 to 0.95mg/mL, a pegylated lipid at or about 0.05 to 0.15mg/mL, a first structural lipid at or about 0.1 to 0.25mg/mL, and a second structural lipid at or about 0.3 to 0.45mg/mL, and further comprising a first buffer at or about 0.01 to 0.09mg/mL, a second buffer at or about 15mg to about 5mg/mL, and a stabilizing agent at or about 50mg to about 15mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition. In a particular embodiment, the lyophilized composition is reconstituted in 0.6 to 0.75ml of salt diluent. The concentration in the lyophilized RSV RNA-LNP composition is determined after reconstitution.

In particular embodiments, the lyophilized RSV RNA-LNP compositions comprise a RSV RNA polynucleotide encoding a RSV polypeptide disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01 to 0.09mg/mL, and more preferably at or about 0.06mg/mL, encapsulated in LNP having a lipid composition of ALC-0315 at or about 0.8 to 0.95mg/mL, ALC-0159 at or about 0.05 to 0.15mg/mL, DSPC at or about 0.1 to 0.25mg/mL, and cholesterol at or about 0.3 to 0.45mg/mL, and further comprising bradykinin at or about 0.01 to about 0.15mg/mL, sucrose at or about 0.65mg/mL, and sucrose at or about 0.5 to about 50 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition. In a particular embodiment, the lyophilized composition is reconstituted in 0.6 to 0.75ml of NaCl diluent (saline). The concentration in the lyophilized RSV RNA-LNP composition is determined after reconstitution.

In some embodiments, the RSV RNA-LNP compositions (prior to lyophilization) comprise the RSV RNA polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01-0.09 mg/mL, encapsulated in LNP having a lipid composition of a cationic lipid at or about 1.0-3.0 mg/mL, a pegylated lipid at or about 0.10-0.35 mg/mL, a first structural lipid at or about 0.4-0.55 mg/mL, a second structural lipid at or about 0.85-1.0 mg/mL, and further comprising a first buffer at or about 0.1-0.3 mg/mL, a second buffer at or about 1.40mg/mL, or about 1-95 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

Thus, in some embodiments, the RSV RNA-LNP compositions (prior to lyophilization) comprise the RSV RNA polynucleotides encoding the RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably at or about 0.01 to 0.09mg/mL, and more preferably 0.15mg/mL, encapsulated in LNP having a lipid composition comprising ALC-0315 at or about 1.0 to 3.0mg/mL, ALC-0159 at or about 0.10 to 0.35mg/mL, DSPC at or about 0.4 to 0.55mg/mL, cholesterol at or about 0.85 to 1.0mg/mL, and further comprising bradykinin at or about 0.1 to about 0.05 mg/mL, sucrose at or about 1 to about 25 mg/mL. In some embodiments, one or more of the foregoing elements may be excluded from the RSV RNA-LNP composition.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises RNA molecules/polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably or about 0.01-0.09 mg/mL, encapsulated in LNP, and further comprises or comprises about 5-15 mM Tris buffer and about 200-400 mM sucrose at a pH of or about 7.0-8.0. In some embodiments, one or more of the foregoing elements may be excluded from the liquid RNA-LNP immunogenic composition.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises RNA molecules/polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably or about 0.01 to 0.09mg/mL, and more preferably or about 0.06mg/mL, encapsulated in LNP, and further comprising or including about 10mM Tris buffer and 300mM sucrose at a pH or about 7.4. In some embodiments, one or more of the foregoing elements may be excluded from the liquid RNA-LNP immunogenic composition.

In some embodiments, the RNA-LNP immunogenic composition (prior to lyophilization) comprises RNA molecules/polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably or about 0.01-0.09 mg/mL, encapsulated in LNP, and further comprises or comprises about 5-15 mM Tris buffer and 200-400 mM sucrose at a pH or about 7.0-8.0, and reconstituted with a 0.9% sodium chloride diluent. In some embodiments, one or more of the foregoing elements may be excluded from the RNA-LNP immunogenic composition.

In some embodiments, the RNA-LNP immunogenic composition (prior to lyophilization) comprises the RNA molecules/polynucleotides encoding RSV polypeptides disclosed herein at a concentration of at least, up to, just below, or between (including or exclusive of) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably or about 0.01-0.09 mg/mL, and more preferably 0.15mg/mL, encapsulated in LNP, and further comprises or comprises about 10mM Tris buffer and 300mM sucrose at a pH or about 7.4, and reconstituted with a 0.9% sodium chloride diluent. In some embodiments, one or more of the foregoing elements may be excluded from the RNA-LNP immunogenic composition.

[ B. vaccine ]

In some embodiments, the pharmaceutical compositions described herein are immunogenic compositions for inducing an immune response. For example, in some embodiments, the immunogenic composition is a vaccine. In some embodiments, the compositions described herein comprise at least one isolated nucleic acid or polypeptide molecule described herein. In a particular embodiment, the immunogenic composition comprises a nucleic acid, and the immunogenic composition is a nucleic acid vaccine. In some embodiments, the immunogenic composition comprises RNA (e.g., mRNA, saRNA), and the vaccine is an RNA vaccine. In other embodiments, the immunogenic composition comprises DNA and the vaccine is a DNA vaccine. In yet other embodiments, the immunogenic composition comprises a polypeptide and the vaccine is a polypeptide vaccine. Conditions and/or diseases that may be treated with the nucleic acid and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or affected by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, and/or inappropriate production of protein or nucleic acid.

In some embodiments, the composition is substantially free of one or more impurities or contaminants and, for example, comprises a nucleic acid or polypeptide molecule of at least, up to, exactly equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% purity or a purity therebetween (inclusive or exclusive), at least 98% purity, or at least 99% purity.

The invention includes methods for preventing, treating and/or ameliorating an infection, disease or condition in an individual comprising administering to the individual an effective amount of an RNA molecule comprising at least one open reading frame encoding a polypeptide or composition described herein. Thus, the present invention encompasses vaccines for both active and passive vaccination embodiments. Immunogenic compositions proposed for use as vaccines can be prepared from RNA molecules encoding one or more polypeptides, such as RSV preF polypeptides. In certain embodiments, the immunogenic composition is lyophilized for easier formulation into a desired vehicle.

The preparation of vaccines containing nucleic acids and/or peptides or polypeptides as active ingredients is generally well known in the art, as exemplified by U.S. Pat. Nos. 4,608,251, 4,601,903, 4,599,231, 4,599,230, 4,596,792, and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared in the form of an injectable preparation in liquid solution or suspension, and solid forms suitable for forming a solution in a liquid or a suspension in a liquid prior to injection may also be prepared. The preparation can also be emulsified. The active immunogenic ingredient is typically admixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and the like, and combinations thereof. In addition, the vaccine may contain a number of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants which enhance the effectiveness of the vaccine, if desired. In a particular embodiment, the vaccine is formulated with a combination of substances, as described in U.S. patent 6,793,923 and 6,733,754, which are incorporated herein by reference. In some embodiments, one or more of the foregoing elements may be excluded from the vaccine.

Vaccines can be administered parenterally, for example, by subcutaneous or intramuscular injection, as is conventional. Additional formulations suitable for other modes of administration include suppositories and in some cases oral formulations. For suppositories, conventional binders and carriers may include, for example, polyalkylene glycols or triglycerides, and such suppositories may be formed from mixtures containing the active ingredient in the range of or about 0.5% to about 10%. In some embodiments, suppositories may be formed from mixtures containing the active ingredient in the range of or about 1% to about 2%. Oral formulations include common excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. In some embodiments, 1,2,3, 4,5 or more of the foregoing excipients may be excluded from the oral formulation. These compositions take the form of solutions, suspensions, lozenges, pills, capsules, sustained-release formulations or powders and contain or contain from about 10% to about 95% of the active ingredient.

Nucleic acid constructs encoding polypeptides and polypeptides may be formulated into vaccines in neutral or salt form. "pharmaceutically acceptable salts" include both acid addition salts and base addition salts. "pharmaceutically acceptable acid addition salts" refer to those salts that retain the bioavailability and properties of the free base, are biologically or otherwise non-adverse, and are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like, and organic acids such as, but not limited to, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylamine sulfonic acid, dodecylsulfuric acid, ethane-1, 2-disulfonic acid, Ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphate, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, Stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing inorganic acids may be excluded. By "pharmaceutically acceptable base addition salt" is meant a salt that retains the biological effectiveness and properties of the free acid, as desired biologically or otherwise. These salts are prepared by addition of an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing inorganic bases may be excluded. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dansyl (deanol), 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine (procaine), sea Zhuo An (hydroabamine), choline, betaine, benzphetamine, benzathine, ethylenediamine, glucosamine, methylreduced glucosamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. In some embodiments, 1,2, 3, 4, 5, or more of the foregoing organic bases may be excluded.

Nucleic acid constructs encoding polypeptides as well as polypeptides or pharmaceutically acceptable salts thereof may contain one or more asymmetric centers and may thus give rise to mirror isomers, non-mirror isomers and other stereoisomeric forms which may be defined as (R) -or (S) -or as (D) -or (L) -for amino acids in terms of absolute stereochemistry. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for preparing/separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Also, all tautomeric forms are intended to be included. "stereoisomers" refers to compounds that are composed of the same atoms bonded by the same bonds but have different three-dimensional structures that are not interchangeable. The present invention encompasses various stereoisomers and mixtures thereof and includes "stereoisomers," which refer to two stereoisomers whose molecules are non-superimposable mirror images of one another. "tautomer" refers to the transfer of a proton from one atom of a molecule to another atom of the same molecule. The present invention discloses tautomers of any of the compounds.

The compounds described herein in free base or acid form may be converted to pharmaceutically acceptable salts thereof by methods known to those skilled in the art using suitable inorganic or organic bases or acids. Salts of the compounds may be converted to their free base or acid forms by standard techniques.

It will be appreciated by those skilled in the art that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amino, mercapto and carboxylic acids. Suitable protecting groups for the hydroxyl group include trialkylsilyl or diarylalkylsilyl groups (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, formamidino and guanidino groups include t-butoxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for mercapto groups include-C (O) -R "(wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters. The protecting group may also be a polymeric resin, such as king resin (WANG RESIN), linke resin (RINK RESIN), or 2-chlorotrityl chloride resin, as will be appreciated by those skilled in the art. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing protecting groups may be excluded. Protecting groups may be added or removed according to techniques known to those skilled in the art (see, e.g., green, t.w. and p.g. m.wutz, protective Groups in Organic Synthesis (1999), 3 rd edition, wiley) and standard techniques described herein.

It will also be appreciated by those skilled in the art that while such protected derivatives of the compounds of the present invention may not possess the same pharmacological activity, they may be administered to a mammal and thereafter metabolized in vivo to form the compounds of the present invention which possess pharmacological activity. Such derivatives may thus be described as "prodrugs". All prodrugs of the compounds of the present invention are included within the scope of the present invention.

Typically, the vaccine is administered in a manner compatible with the dosage formulation, and in an amount such as will be therapeutically effective and immunogenic. The amount to be administered depends on the individual to be treated, including the ability of the individual's immune system to synthesize antibodies and the level of protection desired. The precise amount of active ingredient to be administered will depend upon the discretion of the practitioner. However, a suitable dosage range is approximately hundreds of micrograms of active ingredient per vaccination. The regimen appropriate for the initial administration and booster injection is also variable, but is typically performed for the initial administration followed by subsequent vaccinations and/or other administrations.

The mode of administration can vary widely. Any of the conventional methods for administering vaccines may be applied. These methods are believed to include oral administration within a physiologically acceptable solid matrix or administration by injection or the like in a physiologically acceptable dispersion, rather than enteral administration. The vaccine dose will depend on the route of administration and will vary depending on the size and health of the individual.

In certain embodiments, it will be desirable to administer the vaccine once. In some embodiments, multiple administrations of the vaccine will be required, for example, 2, 3,4, 5, 6 or more administrations. Vaccination may be 1,2, 3,4, 5, 6,7,8 to 5, 6,7,8,9, 10, 11 or 12 weeks apart, including all ranges therebetween. In some embodiments, vaccination may be 1,2, 3,4, 5, 6,7,8,9, 10, 11, or 12 months apart, including all ranges therebetween. Periodic boosting at 1-5 year intervals may be required to maintain the protective level of the antibody.

[ I ] Carrier ]

Pharmaceutically acceptable carriers can include liquid or non-liquid matrices of the composition. If the composition is provided in liquid form, the carrier may be water, such as pyrogen-free water, isotonic saline or buffered (aqueous) solutions, for example phosphate, citrate buffered solutions. Water or buffers containing sodium, calcium and/or potassium salts, such as aqueous buffers, may be used. The sodium, calcium and/or potassium salts may be present in the form of their halides (e.g. chloride, iodide or bromide), in the form of their hydroxides, carbonates, bicarbonates or sulphates etc. Examples of sodium salts include, but are not limited to, NaCl、NaI、NaBr、Na₂CO₃、NaHCO₃、Na₂SO₄、Na₂HPO₄、Na₂HPO₄·₂H₂O, potassium salts include, but are not limited to, KCl, KI, KBr, K ₂CO₃、KHCO₃、K₂SO₄、KH₂PO₄, and examples of calcium salts include, but are not limited to, caCl ₂、CaI₂、CaBr₂、CaCO₃、CaSO₄、Ca(OH)₂. Examples of other carriers may include sugars such as lactose, dextrose, trehalose, and sucrose, starches such as corn starch or potato starch, dextrose, celluloses and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, powdered tragacanth, malt, gelatin, animal fats, solid slip agents such as stearic acid, magnesium stearate, calcium sulfate, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and oils from cocoa, polyols such as polypropylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol, alginic acid. Examples of other carriers may include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents and the pharmaceutical requirements for their use are described in Remington's Pharmaceutical Sciences. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing carriers may be excluded.

[ Ii ] adjuvant ]

Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions. A variety of adjuvants may be used to enhance the antibody response. Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include Freund's adjuvant, oils (such asISA 51), IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, interferon-alpha, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts (such as aluminum hydroxide or other aluminum compounds), MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid a, monophosphoryl lipid a (MPL), lipopeptides (e.g. Pam3 Cys). RIBI, which contains three components extracted from bacteria in a 2% squalene/Tween 80 emulsion, namely MPL, trehalose Dimycolate (TDM) and Cell Wall Skeleton (CWS). Even MHC antigens may be used.

The various methods of achieving adjuvant effects against vaccines include the use of agents such as aluminum hydroxide or aluminum phosphate (alum), which are commonly used in the form of about 0.05% to about 0.1% solutions in phosphate buffered saline, and synthetic sugar polymers used in the form of about 0.25% solutions, respectivelyMixing, and aggregating proteins in the vaccine by heat treatment at a temperature between about 70 ℃ and about 101 ℃ for a period of 30 seconds to 2 minutes. The adjuvant effect can also be produced by coacervation by reactivation with pepsin-treated (Fab) antibodies against albumin, by mixing with bacterial cells, such as Cryptosporidium parvum (C.parvum), endotoxins or lipopolysaccharide components of gram-negative bacteria, by emulsification in physiologically acceptable oil vehicles such as mannitol monooleate (AracelA), or by use of perfluorocarbons as blocking substitutesIs emulsified with a 20% solution of (b).

In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing adjuvants may be excluded.

In addition to adjuvants, it may be desirable to co-administer Biological Response Modifiers (BRMs) to enhance immune responses. BRMs have been shown to up-regulate T cell immunity or down-regulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIMETIDINE) (CIM; 1200 mg/d) (Smith/Kline, PA), or low dose cyclophosphamide (CYP; 300mg/m ²) (Johnson/Mead, NJ) and cytokines such as gamma interferon, IL-2, or IL-12, or genes encoding proteins involved in immune assist functions such as B-7.

[ C ] combination therapy ]

The compositions and related methods of the invention, particularly the administration of RNA molecules encoding RSV preF polypeptides, may also be used in combination with the administration of one or more other therapeutic agents. These include, but are not limited to, administration of traditional therapies, such as antiviral therapies, such as acyclovir (acyclovir), famciclovir (valacyclovir), and famciclovir (famciclovir), or various combinations of antiviral agents. Also included are one or more therapies for treating one or more symptoms of RSV infection, including but not limited to steroids (including corticosteroids), anti-inflammatory agents (including acetaminophen or ibuprofen (ibuprofen)), analgesics, itch relieving creams or lotions, cold compress methods, or various combinations thereof. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing therapeutic agents may be excluded.

Such combination therapies include administration of a single pharmaceutical dosage form of the composition of the invention and one or more additional active agents, and administration of the composition of the invention and each active agent in separate pharmaceutical dosage forms themselves. For example, the compositions of the invention and other active agents may be administered together to a patient in the form of a single dose composition (such as an injection or lozenge or capsule), or each agent may be administered in the form of a separate oral dosage formulation. When separate dosage formulations are used, the compounds of the invention and one or more additional active agents may be administered at substantially the same time (e.g., in parallel) or at separate staggered times (e.g., sequentially), a combination therapy should be understood to include all of these regimens.

In one embodiment, it is contemplated that the vaccine and/or therapy is used in combination with an antiviral treatment. Or the vaccine and/or therapy may be administered at intervals ranging from minutes to weeks before or after treatment with another agent. In embodiments where other agents and/or vaccines are administered separately, it will generally be ensured that a significant period of time has not expired between each delivery, so that the agent and immunogenic composition will still be able to exert a beneficial combined effect on the individual. In such embodiments, it is contemplated that the two modalities may be administered at or about 12-24 hours from each other or about 6-12 hours from each other (e.g., at least, up to, just below, or between any two of (including or exclusive of) 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours from each other. In some cases, it may be desirable to significantly extend the period of administration, with days (2, 3, 4, 5, 6, 7 or longer) to weeks (1, 2, 3, 4, 5, 6, 7, 8 weeks or longer) passing between respective administrations.

Various combinations may be employed, such as an immunogenic polypeptide administered as part of an immunotherapy regimen "a" as:

Administration of the immunogenic compositions of the invention to a patient/individual will follow the general protocol for administration of such compounds, taking into account the toxicity (if any) of the RSV RNA vaccine compositions or other compositions described herein. It is contemplated that the treatment cycle will be repeated as needed. It is also contemplated that different standard therapies (such as moisturizing) may be applied in combination with the described therapies.

[ D ] administration ]

Administration of the compositions described herein may be via any acceptable mode of administration for agents that function similarly. In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intranasally, subcutaneously, intradermally, or intramuscularly. In particular embodiments, the RSV RNA molecule and/or the RNA-LNP composition is administered intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical administration or systemic administration. Systemic administration may include enteral administration (which involves absorption via the gastrointestinal tract) or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the gastrointestinal tract, such as by intravenous injection. In one embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, e.g., for intravenous administration. In some embodiments, 1,2,3, or more of the aforementioned routes of administration may be excluded.

The pharmaceutical compositions may be formulated as preparations in solid, semi-solid, liquid, lyophilized, frozen and/or gaseous forms, such as lozenges, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. In some embodiments, 1,2,3, or more of the foregoing formulations may be excluded. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intranasal, intrasternal injection or infusion techniques. The pharmaceutical compositions described herein are formulated so as to allow the active ingredient contained therein to be bioavailable upon administration of the composition to a patient. The composition to be administered to an individual or patient is in the form of one or more dosage units, where, for example, a lozenge may be a single dosage unit and a container of the compound in aerosol form may hold multiple dosage units. The composition to be administered will in any case contain a therapeutically and/or prophylactically effective amount of a compound within the scope of the present invention, or a pharmaceutically acceptable salt thereof, to treat the disease or condition of interest in accordance with the teachings described herein.

Pharmaceutical compositions within the scope of the invention may be in solid or liquid form and may be frozen or lyophilized. In one embodiment, the one or more carriers are microparticles, such that the composition is in the form of, for example, a lozenge or powder. The carrier or carriers can be liquid such that the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable for, for example, inhalation administration. In some embodiments, when intended for oral administration, the pharmaceutical composition is in solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms included within the forms considered herein as solid or liquid.

As solid compositions for oral administration, the pharmaceutical compositions may be formulated as powders, granules, compressed lozenges, pills, capsules, chewable tablets, caplets or the like. Such solid compositions will typically contain one or more inert diluents or edible carriers. Additionally, one or more of binders such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin, excipients such as starch, lactose or dextrin, disintegrants such as alginic acid, sodium alginate, and the like, may be present or excluded,Corn starch and the like, lubricants, such as magnesium stearate or the likeSlip agents such as colloidal silica, sweeteners such as sucrose or saccharin, flavoring agents such as peppermint, methyl salicylate or orange flavoring, and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain a liquid carrier such as polyethylene glycol or oil in addition to the above types of substances. In some embodiments, 1,2, 3 or more of the foregoing elements may be excluded from the solid composition.

The pharmaceutical compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions or suspensions. The liquid may be used for oral administration or for delivery by injection, to name two examples. In some embodiments, when intended for oral administration, the compositions contain, in addition to the compounds of the present invention, one or more of sweeteners, preservatives, dyes/colorants and flavoring agents. In compositions intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may be included or excluded.

Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may include or exclude one or more of adjuvants such as sterile diluents such as water for injection, saline solutions (e.g., physiological saline), ringer's solution, isotonic sodium chloride, fixed oils (such as synthetic mono-or diglycerides, which may act as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol or other solvents, antibacterial agents such as benzyl alcohol or methyl parahydroxybenzoate, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediamine tetraacetic acid, buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose, agents such as sucrose or trehalose which act as cryoprotectants. Parenteral formulations may be packaged in ampules, disposable syringes or multiple dose vials made of glass or plastic. In one embodiment, physiological saline is an adjuvant. In one embodiment, the injectable pharmaceutical composition is sterile. Liquid pharmaceutical compositions intended for parenteral or oral administration should contain a certain amount of the compound such that a suitable dosage will be obtained.

The pharmaceutical composition may include various materials that regulate the physical form of the solid or liquid dosage unit. For example, the composition may include a material that forms a coating shell around the active ingredient. The material forming the coating shell is generally inert and may be, for example, sugar, shellac or other enteric coating agents.

The pharmaceutical composition may comprise dosage units that may be administered in aerosol form. The term aerosol means a variety of systems ranging from those of a colloidal nature to those composed of pressurized packages. Delivery may be by liquefied or compressed gas or by a suitable pump system that dispenses the active ingredient. Aerosols of the compounds may be delivered in single-phase, biphasic or triphasic systems in order to deliver one or more active ingredients. The delivery of the aerosol includes the necessary containers, activators, valves, sub-containers, etc., which together may form a kit. The skilled artisan can determine the preferred aerosols without undue experimentation.

The pharmaceutical compositions may be prepared by methods well known in the pharmaceutical arts. For example, a pharmaceutical composition intended for administration by injection may be prepared by combining a nucleic acid or polypeptide with sterile distilled water or other carrier so as to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a compound in accordance with the teachings herein in order to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.

The pharmaceutical composition according to the invention or a pharmaceutically acceptable salt thereof is generally administered in a "therapeutically effective amount" or a "prophylactically effective amount" and in a "pharmaceutically acceptable formulation". The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active components of the pharmaceutical composition. The term "therapeutically effective amount" and "prophylactically effective amount" refers to an amount that alone or in combination with other dosages achieves the desired response or desired effect. In the case of treating a particular disease, in one embodiment, the desired response involves inhibiting the course of the disease. This includes slowing disease progression, and in particular interrupting and/or reversing disease progression. The desired response to treating a disease may also be to delay the onset of the disease or condition and/or to prevent the onset of the disease or condition.

Compositions within the scope of the present invention are administered in a therapeutically and/or prophylactically effective amount that will vary depending on a variety of factors, including the activity of the particular therapeutic and/or prophylactic agent employed, the metabolic stability and length of action of that therapeutic and/or prophylactic agent, the individual parameters of the patient, including the age, weight, general health, sex, and diet of the patient, the mode, time, and/or duration of administration, the rate of secretion, the pharmaceutical combination, the severity of the particular disorder or condition, and the ongoing therapy of the individual. In some embodiments, 1, 2, 3, 4, 5 or more of the factors may be excluded from determining a therapeutically and/or prophylactically effective amount. Thus, the dosage of the compositions described herein to be administered can depend on a variety of such parameters. In cases where the response in the patient is insufficient at the initial dose, a higher dose (or effectively higher dose achieved by another more local route of administration) may be used. In some embodiments, the composition (e.g., RSV RNA-LNP composition) may be administered at a dosage level sufficient to deliver an amount of 0.0001ng/μg/mg～100ng/μg/mg、0.001ng/μg/mg～0.05ng/μg/mg、0.005ng/μg/mg～0.05ng/μg/mg、0.001ng/μg/mg～0.005ng/μg/mg、0.05ng/μg/mg～0.5ng/μg/mg、0.01ng/μg/mg～50ng/μg/mg、0.1ng/μg/mg～40ng/μg/mg、0.5ng/μg/mg～30ng/μg/mg、0.01ng/μg/mg～10ng/μg/mg、0.1ng/μg/mg～10ng/μg/mg、 or 1ng/μg/mg to 25ng/μg/mg per kilogram body weight of the individual per day, one or more times a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, prophylactic, and/or imaging effect (see, e.g., the unit dosage ranges described in International publication No. WO2013/078199, which is incorporated herein by reference in its entirety). In some embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a dosage level sufficient to deliver at least, up to, just below, or between any two (inclusive or exclusive) 0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100ng/μg/mg per kilogram of body weight of the individual per day, one or more times a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, prophylactic, and/or imaging effects.

In some embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or in total between any two of (inclusive or exclusive) or at a dosage level sufficient to deliver at least, up to, just below, or in total between any two of 0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100ng/μg/mg per day, one or more times per day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, prophylactic, and/or imaging effects.

In particular embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or in total between any two of the following (inclusive or exclusive) or at a dosage level sufficient to deliver at least, up to, just below, or in total between any two of the following, ：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100mg/mL of RSV RNA encapsulated in the LNP.

In exemplary embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or at a dosage level between (including or exclusive of) any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL of RSV RNA encapsulated in LNP. In exemplary embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or at a dosage level between (inclusive or exclusive of) any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg of RSV RNA encapsulated in LNP.

In particular embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or in total between any two of the following (inclusive or exclusive) or at a dosage level sufficient to deliver at least, up to, just below, or in total between any two of the following (inclusive or exclusive) ：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100 μg/mL of RSV RNA encapsulated in the LNP.

In exemplary embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or at a dosage level between (including or exclusive of) any two of 1, 15, 30, 45, 60, 75, 90, 100, or more micrograms/milliliter of RSV RNA encapsulated in LNP. In exemplary embodiments, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at least, up to, just below, or at a dosage level between (including or exclusive of) any two of 1, 15, 30, 45, 60, 75, 90, 100, or more micrograms of RSV RNA encapsulated in LNP.

The desired dose may be delivered multiple times a day (e.g., 1,2, 3,4,5, or more times a day), every other day, every third day, weekly, biweekly, every third week, every fourth week, every 2 months, every third month, every 6 months, or each year, etc. In certain embodiments, the desired dose may be delivered using a single dose administration. In certain embodiments, the desired dose may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, twelve, thirteen, fourteen or more administrations). When multiple applications are employed, a split application regimen may be used. The time of administration between the initial administration of the composition and the subsequent administration of the composition 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 some embodiments, the composition (e.g., RSV RNA-LNP composition) can be administered in a single dose. In some embodiments, the composition (e.g., RSV RNA-LNP composition) can be administered twice (e.g., on day 0 and after or at about day 7, day 0 and after or at about day 14, day 0 and after or at about day 21, day 0 and after or at about day 28, day 0 and after or at about day 60, day 0 and after or at about day 90, day 0 and after or at about day 120, day 0 and after or at about day 150, day 0 and after or at about day 180, day 0 and after or at about month 1, day 0 and after or at about month 2, day 0 and after or at about month 3, day 0 and after or at about month 6, day 0 and after or at about month 9, day 0 and after or at about month 12, day 0 and after or at about month 18, day 0 and after or at about year 2, day 0 and after or at about month, day 2, and after about year 5, wherein the total dose of total RNA(s) is delivered at least between total dose(s) of 5, including at least one of 5g and/or more of total of 5g (including total or more of these doses). The present invention encompasses higher and lower dosages and frequencies of administration. For example, a composition (e.g., an RSV RNA-LNP composition) can be administered three or four times.

Periodic boosting at 1-5 year intervals may be required to maintain the protective level of the antibody. As used herein, the term "boost" refers to the additional administration of a composition (e.g., RSVRNA-LNP composition). The boost may be administered after early administration of the composition.

In some embodiments, the composition (e.g., RSV RNA-LNP composition) is administered ：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100ng/μg/mg of RSV RNA encapsulated in LNP to the individual in a single dose, at least, up to, just below, or between any two of the following (inclusive or exclusive). In some embodiments, the composition (e.g., RSV RNA-LNP composition) is administered to the individual in a single dose of at least, up to, just below, or between any two of (inclusive or exclusive) 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or higher dose of RSV RNA encapsulated in LNP.

In some embodiments, the composition (e.g., RSV RNA-LNP composition) is administered ：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、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、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、99 or 100ng/μg/mg of RSV RNA encapsulated in LNP to the individual in at least, up to, just below, or in between any two of the following (inclusive or exclusive) two doses. In some embodiments, the composition (e.g., RSV RNA-LNP composition) is administered to the individual in at least, up to, just below, or between any two of (inclusive or exclusive) two doses of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or higher doses of RSV RNA encapsulated in LNP.

In particular embodiments, a composition (e.g., an RSV RNA-LNP composition) can be administered without administration of two times (e.g., on day 0 and 28, on day 0 and 60, on day 0 and 180, after day 0 and 2 months, after day 0 and 6 months, after day 0 and one year, etc.), wherein each administration is at least, up to, just below, or a total dose between any two of (including or exclusive) or a dose level sufficient to deliver at least, up to, just below, or a total dose between any two of (including or exclusive) the following: RSV RNA encapsulated in LNP at doses of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or higher.

[ IX. methods of use ]

Provided herein are compositions (e.g., pharmaceutical compositions comprising RSV RNA molecules and/or RSV RNA-LNP), methods, kits, and reagents for preventing and/or treating RSV in humans and other mammals.

RSV RNA compositions (e.g., RSV RNA-LNP compositions) are useful as therapeutic and/or prophylactic agents. It can be used in medicine for preventing and/or treating infectious diseases. In an exemplary embodiment, the RSV RNA composition is used to provide prophylactic protection against acute lower respiratory tract infection (ALRI) of any genotype, strain, or isolate. It is envisaged that there may be circumstances in which a person is at risk of infection with more than one RSV strain. RSV RNA compositions (e.g., RSV RNA-LNP compositions) are particularly suited for combination vaccination routes due to a variety of factors including, but not limited to, manufacturing speed, ability to rapidly adjust vaccines to accommodate perceived geographic threats, and the like. Furthermore, because RSV RNA compositions (e.g., RSV RNA-LNP compositions) use humans to produce antigenic proteins, RSV RNA compositions (e.g., RSV RNA-LNP compositions) are suitable for producing larger, more complex antigenic proteins, allowing for proper folding, surface expression, antigen presentation, etc. in a human individual. To protect against more than one RSV strain, a combination RSV RNA composition can be administered that includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a first RSV and further includes RNA encoding at least one antigenic polypeptide protein (or antigenic portion thereof) of a second RSV. The RSV vaccines of the invention are useful for preventing RSV (infection-related disorders, including pneumonia and bronchitis), and are particularly useful for preventing and/or treating immune dysfunction and elderly patients to prevent or reduce the severity and/or duration of RSV infection.

In some embodiments, the RSV RNA compositions (e.g., RSV RNA-LNP compositions) of the invention are administered to an individual (e.g., a mammalian individual, such as a human individual) and the RNA polynucleotide is translated in vivo to produce the antigenic polypeptide. RSV RNA compositions (e.g., RSV RNA-LNP compositions) can be induced to translate a polypeptide (e.g., an antigen or immunogen) in a cell, tissue, or organism. In the exemplary embodiment, such translation occurs in vivo, but embodiments are contemplated in which such translation occurs ex vivo, in culture, or in vitro. In an exemplary embodiment, a cell, tissue or organism is contacted with an effective amount of an RSV RNA composition (e.g., an RSV RNA-LNP composition) that includes an RNA molecule having at least one translatable region encoding an antigenic polypeptide (e.g., an RSV antigen).

In some embodiments, the RSV RNA compositions of the invention can be used to sensitize immune effector cells, such as Peripheral Blood Mononuclear Cells (PBMCs) that are activated ex vivo, and then infused (re-infused) into an individual.

In some embodiments, at least a portion of the RNA is delivered to the target cell after administration of the RSV RNA molecules described herein (e.g., formulated as RNA-LNP).

In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce the polypeptide or protein encoded thereby. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell in the spleen, such as a professional antigen presenting cell. In some embodiments, the target cell is a dendritic cell and/or a macrophage. RNA molecules (such as the RNA-LNP described herein) can be used to deliver RNA to such target cells. Thus, the invention also relates to a method for delivering RNA to a target cell in an individual comprising administering to the individual an RNA particle as described herein.

In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce a polypeptide or protein encoded by the RNA. "coding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have defined nucleotide sequences (e.g., rRNA, tRNA and mRNA) or defined amino acid sequences and biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand whose nucleotide sequence corresponds to the mRNA sequence and which is normally provided in the sequence listing, and the non-coding strand which serves as a template for transcription of a gene or cDNA, may both be referred to as a protein or other product encoding the gene or cDNA.

In some embodiments, a nucleic acid composition described herein (e.g., a composition comprising RSV RNA-LNP) is characterized by an induced and/or enhanced immune response that varies with antigen in a cell (e.g., when administered to an individual). The increased antigen production may be demonstrated, for example, by increased cell transfection (percentage of cells transfected with an RNA vaccine), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated by increased duration of protein translation from the modified polynucleotide), and/or altered antigen-specific immune responses of the host cell.

In some embodiments, the invention relates to a method of inducing an immune response against RSV in an individual. The method comprises administering to the individual an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein to generate an immune response against RSV.

In another embodiment, the invention relates to a method of vaccinating an individual. The method comprises administering to an individual in need thereof an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another embodiment, the invention relates to a method of treating and/or preventing infectious diseases. The method comprises administering to the individual an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein.

In another embodiment, the invention relates to a method of treating and/or preventing RSV infection and/or a condition caused by RSV and/or reducing the severity thereof. The method comprises administering to the individual an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein.

In another embodiment, the invention relates to a method of treating and/or preventing an infectious disease and/or reducing the severity of an infectious disease in an individual, e.g., by inducing an immune response against the infectious disease in the individual. In some embodiments, the method comprises administering a priming composition comprising an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein, and administering a boosting composition comprising an effective amount of an RNA molecule, RNA-LNP, and/or composition. In some embodiments, the composition elicits an immune response, including an antibody response. In some embodiments, the composition elicits an immune response, including a T cell response and/or a B cell response. In some embodiments, the immune response comprises a T cell response and a B cell response. In some embodiments, the composition elicits a neutralizing immune response. The neutralizing immune response is an immune response that is a neutralizing antibody response and/or an effective neutralizing T cell response. In some embodiments, the neutralizing antibody reaction produces an antibody level that meets or exceeds a seroprotection threshold. In some embodiments, the composition elicits an effective T cell response. The effective T cell response is a response that produces baseline levels of infectious disease activated and/or infectious disease specific T cells, including CD8 ⁺ and CD4 ⁺ T helper type 1 cells. In some embodiments, the effective T cells comprise a high proportion of CD8 ⁺ T cells and/or CD4 ⁺ T cells relative to the baseline level (in untreated individuals). In some embodiments, these T cells differentiate towards an early differentiated memory phenotype that coexpresses CD27 and CD 28.

In another embodiment, the invention relates to a method of treating and/or preventing RSV infection and/or a condition caused by RSV and/or reducing the severity thereof in an individual, e.g., by inducing an immune response against RSV in the individual. In some embodiments, the method comprises administering a priming composition comprising an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein, and administering a boosting composition comprising an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein. In some embodiments, the composition elicits an immune response, including an antibody response. In some embodiments, the composition elicits an immune response, including a T cell response and/or a B cell response. In some embodiments, the immune response comprises a T cell response and a B cell response. In some embodiments, the composition elicits a neutralizing immune response. The neutralizing immune response is an immune response that is a neutralizing antibody response and/or an effective neutralizing T cell response. In some embodiments, the neutralizing antibody reaction produces an antibody level that meets or exceeds a seroprotection threshold. In some embodiments, the composition elicits an effective T cell response. The effective T cell response is a response that produces baseline levels of infectious disease activated and/or infectious disease specific T cells, including CD8 ⁺ and CD4 ⁺ T helper type 1 cells. In some embodiments, the effective T cells comprise a high proportion of CD8 ⁺ T cells and/or CD4 ⁺ T cells relative to the baseline level (in untreated individuals). In some embodiments, these T cells differentiate towards an early differentiated memory phenotype that coexpresses CD27 and CD 28.

The methods disclosed herein can involve administering to an individual an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide, thereby inducing an immune response in the individual specific for the RSV antigenic polypeptide, wherein the anti-antigenic polypeptide antibody titer in the individual increases after vaccination relative to a vaccination prophylactically effective dose (e.g., a therapeutically effective dose at a clinically acceptable level to prevent infection with a virus) of an anti-antigenic polypeptide antibody titer in an individual of a traditional vaccine against RSV. An "anti-antigenic polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide. In some embodiments, the anti-antigenic polypeptide antibody titer in the individual is increased or not increased by an amount of at least, up to, between (including or exclusive of) or just below any two of the following, 1,2,3, 4, 5, 6,7,8, 9 or 10 logs after administration of the RSV RNA-LNP composition, relative to the anti-antigenic polypeptide antibody titer in an individual administered a prophylactically effective dose of a traditional composition against RSV (e.g., a standard care dose of a recombinant or purified RSV protein vaccine, an attenuated or inactivated RSV vaccine or an RSV VLP vaccine). In some embodiments, the anti-antigenic polypeptide antibody titer in the individual is increased or not increased by an amount of at least, up to, between (including or exclusive of) or just below any two of the following, 1,2,3, 4, 5, 6,7,8, 9, 10, 100 or 1000 fold after administration of the RSV RNA-LNP composition relative to the anti-antigenic polypeptide antibody titer in an individual administered a prophylactically effective dose of a traditional composition against RSV (e.g., a standard care dose of a recombinant or purified RSV protein vaccine, an attenuated or inactivated RSV vaccine, or an RSV VLP vaccine).

In some embodiments, an effective amount of an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide results in a 2-fold to 200-fold (e.g., at least, up to, just below, or between (including or exclusive of) any two of 2,3, 4,5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200-fold) increase in serum neutralizing antibodies to RSV relative to a traditional composition (e.g., standard care dose of recombinant or purified RSV protein vaccines, attenuated or inactivated RSV vaccines, or RSV VLP vaccines) to RSV.

In some embodiments, the effective amount of the RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide is a dose that is at least 2-fold reduced equivalent to a standard care dose of a traditional composition for RSV. For example, an effective amount of an RSV RNA-LNP composition may or may not be at least a reduced dose of 2,3, 4, 5, 6,7, 8, 9, 10, 20, 50, 100, 250, 500, or 1000 times equivalent to the standard care dose for a traditional composition of RSV. In some embodiments, the anti-RSV antigenic polypeptide antibody titers produced in individuals administered an effective amount of an RSV RNA-LNP composition are equivalent to the anti-RSV antigenic polypeptide antibody titers produced in control individuals administered a standard care dose of a traditional composition against RSV. In some embodiments, the effective amount of the RSV RNA-LNP composition is or is not a dose that is 2-fold to 1000-fold reduced (e.g., at least, up to, just below, or between any two of the following (including an exclusive )：2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、1280、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、4360、470、480、490、500、510、520、530、540、550、560、5760、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990 or 1000-fold reduction) equivalent to the standard care dose of the traditional composition for RSV, wherein the anti-RSV antigenic polypeptide antibody titer produced in the individual is equivalent to the anti-RSV antigenic polypeptide antibody titer produced in a control individual administered the standard care dose of the traditional composition for RSV.

As used herein, a traditional composition for RSV refers to a composition other than the RNA molecules, RNA-LNPs, and/or compositions described herein. For example, traditional compositions include, but are not limited to, live microbial vaccines, killed microbial vaccines, attenuated vaccines, subunit vaccines, protein antigen vaccines containing recombinant proteins produced in heterologous expression systems or purified from a large number of pathogenic organisms, DNA vaccines, virus-like particle (VLP) vaccines containing viral capsid proteins (e.g., pre-and/or post-fusion F proteins) but lacking viral genomes, and the like. In an exemplary embodiment, the traditional vaccine is a vaccine that has been approved for regulatory approval and/or registered by a national drug administration, such as the U.S. food and drug administration (Food and Drug Administration; FDA) or European drug administration (European MEDICINES AGENCY; EMA). As provided herein, "standard care" refers to medical or psychiatric guidelines and may be general or specific. "Standard care" prescribes appropriate treatment based on cooperation between medical professionals who are scientific evidence and involved in the treatment of a given condition. It is the diagnosis and treatment method that a physician/clinician should follow for a certain type of patient, disorder or clinical situation. As provided herein, "standard care dose" refers to the dose of a traditional composition for RSV that a physician/clinician or other medical professional will administer to an individual to treat and/or prevent RSV or an RSV-related condition while following standard care guidelines for treating and/or preventing RSV or an RSV-related condition.

In some embodiments, the RNA molecules, RNA-LNPs, and/or compositions described herein (e.g., RSV RNA-LNP compositions comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide) produce prophylactically and/or therapeutically effective levels, concentrations, and/or titers of antigen-specific antibodies in the blood or serum of an individual. As defined herein, the term antibody titer refers to the amount of antigen-specific antibody produced in an individual (e.g., a human individual). In an exemplary embodiment, antibody titers are expressed as the reciprocal of the maximum dilution (in serial dilutions) that still yields a positive result. In an exemplary embodiment, the antibody titer is determined or measured by an enzyme-linked immunosorbent assay (ELISA). In an exemplary embodiment, the antibody titer is determined or measured by a neutralization assay, e.g., by a micro-neutralization assay. In certain embodiments, the antibody titer measurements are expressed as ratios, such as 1:40, 1:100, and the like.

In exemplary embodiments, effective RNA molecules, RNA-LNPs, and/or compositions described herein (e.g., RSV RNA-LNP compositions comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide) produce an antibody titer of greater than 1:10, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:5000, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary embodiments, the antibody titer is produced or reached 10 days after vaccination, 20 days after vaccination, 30 days after vaccination, 40 days after vaccination, or 50 days or more after vaccination. In an exemplary embodiment, the potency is generated or reached after a single dose of vaccine administered to the individual. In other embodiments, the potency is generated or reached after multiple doses, e.g., after a first and second dose (e.g., a boost dose).

The methods disclosed herein can involve administering to an individual an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide, thereby inducing an immune response in the individual that is specific for the RSV antigenic polypeptide, wherein the immune response in the individual is equivalent to an immune response in an individual administered a traditional composition directed against RSV, which is or is not at least below, at most below, between any two of the following (inclusive or exclusive) or at a dosage level ：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98 or 100 times the amount just below, relative to the RNA composition.

In some embodiments, the RNA molecule, RNA-LNP and/or composition is used as a vaccine. In some embodiments, the RNA molecules, RNA-LNPs, and/or compositions can be used in various therapeutic or prophylactic methods for preventing, treating, or ameliorating acute lower respiratory tract infections (ALRI) or conditions associated with respiratory tract disorders, including pneumonia and bronchitis.

In some embodiments, the RNA molecules, RNA-LNP, and/or compositions can be used in various therapeutic or prophylactic methods for preventing, treating, or ameliorating acute lower respiratory tract infections (ALRI), including pneumonia and bronchitis.

In some embodiments, the methods of the invention relate to prognosis, diagnosis, testing, monitoring, and/or treatment of an individual suspected of having, being at risk of having (e.g., RSV) and/or having symptoms of (e.g., RSV). The individual may be suffering from one or more symptoms of an infectious disease (e.g., RSV). One or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., RSV) of an individual can be tested by one or more diagnostic tests (e.g., PCR tests for detection of RSV in skin lesions; tzanck smears; igM serum tests; ELISA for IgG detection, glycoprotein-based ELISA, latex agglutination and/or indirect fluorescent antibodies; direct fluorescent antibody analysis, virus culture, etc.). In some embodiments, an individual who has, has been at risk of having, has (has been at risk of having) an infectious disease (e.g., RSV), has (has) a symptom of an infectious disease (e.g., RSV) is prognosis, diagnosis, monitoring and/or treatment of an infectious disease (e.g., RSV) by one or more diagnostic tests (e.g., PCR test for detecting RSV; tzanck smear; igM serum test; ELISA for IgG detection, glycoprotein-based ELISA, latex agglutination and/or indirect fluorescent antibodies; direct fluorescent antibody analysis, virus culture, etc.) based on the measurement, analysis or detection of one or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., RSV) in a sample from the individual (e.g., blood, saliva, tissue, bone, muscle, cartilage and/or skin).

The RSV RNA compositions can be administered prophylactically to healthy individuals, or early in the infection during latency or during active infection after onset of symptoms. In some embodiments, the individual has immunocompetence. In some embodiments, the individual is immunocompromised.

In some embodiments, the RNA molecule, RNA-LNP, and/or composition is administered in a single dose. In some embodiments, a second, third, or fourth dose may be administered. In some embodiments, the RNA molecule, RNA-LNP and/or composition is administered in multiple doses.

In some embodiments, the RNA molecule, RNA-LNP, and/or composition is administered Intramuscularly (IM) or Intradermally (ID).

The invention further provides a kit comprising an RNA molecule, an RNA-LNP and/or a composition.

In some embodiments, the RNA molecules, RNA-LNPs, and/or compositions described herein are administered to an individual less than about 1 year old, or about 1 year old to about 10 years old, or about 10 year old to about 20 years old, or about 20 year old to about 50 years old, or about 60 year old to about 70 years old, or more.

In some embodiments, an individual is at least, at most, exactly, or between any two of the years less than 1 year old, greater than 5 years old, greater than 10 years old, greater than 20 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, or older. In some embodiments, the individual is older than 50 years.

In some embodiments, the individual is at least, at most, just below, or between (inclusive or exclusive) any two of about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. In some embodiments, the individual may be about 50 years old or older.

In some embodiments, the individual is at least, at most, just below, or between (inclusive or exclusive) any two of age 1 year old or older, 5 years old or older, 10 years old or older, 20 years old or older, 30 years old or older, 40 years old or older, 50 years old or older, 60 years old or older, 70 years old or older, or older. In some embodiments, the individual may be 50 years old or older.

[ X. clinical study ]

The RSV RNA-LNP vaccine of the invention comprises a nucleoside modified mRNA (modified RNA; modRNA) encoding a pre-fusion F (preF) polypeptide from RSV. The RSV RNA-LNP vaccine can comprise RNA that comprises a single-stranded, 5' capped, and polyadenylation modified RNA that is translated after entry into a cell. The RNA comprises an Open Reading Frame (ORF) encoding a variant of the RSV preF polypeptide. Furthermore, as described herein, the RNA can comprise structural components, such as untranslated regions (UTRs), that are optimized for high efficacy of the RNA. RSV RNA-LNP can comprise an RNA provided in table 5 of example 1 disclosed herein. The RSV RNA-LNP may comprise the RNAs provided in tables 1-3 of example 6 disclosed herein. The RNA may also contain a substitution of uridine by 1-methyl-pseudouridine to reduce recognition of vaccine RNA by innate immune sensors such as the toll-like receptors (TLRs) 7 and 8, resulting in reduced innate immune activation and increased protein translation.

The RNA molecules described herein are formulated/encapsulated IN Lipid Nanoparticles (LNPs) to enable delivery of RNA into host cells after, for example, intramuscular (IM), intradermal (ID), or Intranasal (IN) injection. The LNP formulation may comprise two functional lipids (ALC-0315 and ALC-0159) and two structural lipids (DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine) and cholesterol). In some embodiments, 1,2, 3, or more of the foregoing lipids may be excluded from the LNP formulation. The efficacy of RNA vaccines is optimized by LNP encapsulation, which protects RNA from extracellular RNase degradation and facilitates delivery in cells. After IM injection of RSV RNA-LNP vaccine, LNP is taken up by the cells and RNA is released into the cytosol. In the cytosol, the RNA is translated and the encoded viral antigen is produced.

The examples herein demonstrate that the RSV RNA-LNP vaccine of the invention is immunogenic in mice and induces both humoral and cell-mediated immune responses in mice.

Clinical studies of the invention assess the safety, tolerability and immunogenicity of RSV RNA-LNP vaccines against RSV. For example, RSV RNA-LNP vaccines may be indicated for active immunization to prevent acute lower respiratory tract infections (ALRI) in adults (e.g.,. Gtoreq.45,. Gtoreq.50,. Gtoreq.55,. Gtoreq.60,. Gtoreq.70 years, etc., or 50-69 years), including pneumonia and bronchitis caused by RSV. The RSV RNA-LNP vaccine can be administered at different one or more of the dose levels, dose formulations, number of doses, and time of administration described herein, including but not limited to:

single dose schedule or double dose schedule (e.g. day 0 and after or about 2 months, or day 0 and after or about 6 months)

At different dosage levels (e.g. at or about 15 μg, 30 μg, 60 μg, 90 μg, 100 μg or higher per administration)

In different formulations (not lyophilized and/or lyophilized)

RSV RNA-LNP can be presented as a liquid or lyophilized formulation. The administration of the RSV RNA-LNP vaccine may or may not be in the range of about 15 μg, 30 μg, 60 μg, 90 μg, 100 μg or higher per dose, with an injection volume of or about 0.25-1 mL (e.g., or about 0.25, 0.5, 1 mL). Dilution with sterile 0.9% sodium chloride (normal saline) may be required.

Targets for RSV RNA-LNP clinical studies may include, but are not limited to:

describing the safety and tolerability profile of RSV RNA-LNP vaccine administered at selected dose levels and schedules in participants.

Describing the immune response elicited in the participants by commercially available vaccines against RSV and RSV RNA-LNP vaccines administered at selected dose levels and times.

In some embodiments, the RNA molecules, RNA-LNPs, and compositions thereof disclosed herein encoding RSV polypeptides have a efficacy (or effectiveness) against RSV of greater than or no greater than 50% (e.g., at least, up to, just below, or between any two of: 50%, 60%, 70%, 80%, 90%, or more). Standard analysis can be used to assess vaccine efficacy (see, e.g., weinberg et al, J effect Dis.2010, 1/6; 201 (11): 1607-10). For example, vaccine efficacy may be measured by double blind, randomized, clinically controlled trials, such as those described herein. Vaccine efficacy can be expressed as a proportional decrease in disease invasion rate (AR) between the unvaccinated (ARU) and vaccinated (ARV) study groups, and can be calculated from the Relative Risk (RR) of disease in the vaccinated group using the following formula:

efficacy = (ARU-ARV)/aru×100, and

Efficacy= (1-RR) ×100.

Likewise, standard assays can be used to assess vaccine effectiveness (see, e.g., weinberg et al JInfect Dis.2010, 1 month; 201 (11): 1607-10). Vaccine effectiveness is an assessment of how a vaccine (which may have demonstrated high vaccine efficacy) reduces disease in a population. This measurement can assess the net balance of benefits and adverse effects of the vaccination program, rather than just the vaccine itself, under natural field conditions, rather than in controlled clinical trials. Vaccine effectiveness is proportional to vaccine efficacy (potency), but is also affected by the extent of target group vaccination in the population and other non-vaccine related factors that affect the actual outcome of hospitalization, dynamic interrogation and/or cost. For example, retrospective case control analysis may be used, in which vaccination rates among a set of infected cases and appropriate controls are compared. Vaccine effectiveness can be expressed as poor, using the Odds Ratio (OR) of infection that still occurs after vaccination:

effectiveness= (1-OR) ×100.

In some embodiments, the efficacy of the RSV polypeptide, RNA-LNP, and compositions thereof is at least 60% relative to a control individual not vaccinated. For example, the efficacy can be at least, up to, just below, or between any two (inclusive or exclusive) of 65%, 70%, 75%, 80%, 85%, 95%, 98%, or 100% relative to a control individual not vaccinated.

[ Example ]

The following are examples of specific embodiments for practicing the invention. The following examples are included to demonstrate embodiments of the invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should appreciate that, in accordance with the invention, many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should, of course, be allowed for.

Example 1 production of RSV preF modRNA constructs

The RNA constructs produced herein encode RSV F protein wild-type (WT) and RSV F protein variants/mutants (i.e., RSV pre-fusion F protein). Table 4 shows the WT F protein (WT F) and variant RSV preF proteins.

[ Table 4.RSV F protein, description ]

DNA sequences encoding RSV F proteins were prepared and used in an in vitro transcription reaction to produce RNA. In vitro transcription of RNA is known in the art and described herein. The DNA templates were cloned into plasmid vectors with backbone sequence components (T7 promoter, 5 'and 3' utr, poly a tail) for improved RNA stability and translation efficiency. DNA was purified, quantified spectrophotometrically, and transcribed in vitro by T7 RNA polymerase in the presence of trinucleotide cap 1 analog ((m ₂ ^7,3′-O)Gppp(m^2′-O) ApG) (TriLink), and wherein N1-methyl pseudouridine (ψ) replaces uridine (modified RNA (modRNA)).

RSV RNA is produced from Codon Optimized (CO) DNA to achieve stability and good protein expression. Table 5 shows RNA constructs and corresponding sequences of the invention comprising a 5'UTR, an open reading frame encoding a Respiratory Syncytial Virus (RSV) polypeptide, a 3' UTR, and a multi-A tail.

[ Table 5.RSV F mod RNA construct ]

* The 5'UTR sequence includes a 5' cap sequence

* The poly-A tail length may contain +1/-1A

Example 2 production of RSV PREF SARNA constructs

SaRNA synthesis is performed via In Vitro Transcription (IVT) and purified by ultrafiltration/diafiltration (UFDF-1). Next, the saRNA is enzymatically capped and purified by chromatography and final UFDF-2, followed by final filtration and dispensing.

TABLE 6 RSV F847A saRNA construct (encoding the RSV F protein having SEQ ID NO: 4) comprising the 5' cap-5 ' UTR-nsP1-nsP2-nsP3-nsP 4-subgenomic promoter-RSV [ ORF ] -3' UTR-multi A tail in order ]

TABLE 7 RSV F847B saRNA construct (encoding the RSV F protein having SEQ ID NO: 6) comprising the 5' cap-5 ' UTR-nsP1-nsP2-nsP3-nsP 4-subgenomic promoter-RSV [ ORF ] -3' UTR-multi-A tail in order ]

RNA constructs	Start [ SEQ ID NO:16]	Termination [ SEQ ID NO:16]	SEQ ID NO
				5'-CAP	1	1	-
5'-UTR	2	45	52
				NSP1	46	1650	55
NSP2	1651	4032	56
				NSP3	4033	5703	57
NSP4	5704	7527	58
				Subgenomic promoters	7528	7562	54
RSV[ORF]	7563	9290	12
				3'-UTR	9291	9407	24
Multiple A tails	9408	9487	26

EXAMPLE 3 preparation of RSV PREF RNA formulated in LNP

The LNP formulation contains 2 functional lipids ALC-0315 and ALC-0159, and 2 structural lipids DSPC (1, 2-distearoyl-sn-glycero-3-phosphorylcholine) and cholesterol. The physicochemical properties and structures of the 4 lipids are shown in table 8 below.

Lipid nanoparticles were prepared and tested according to the general procedure described in U.S. patent 9737619 (PCT publication No. WO 2015/199952) and U.S. patent 10166298 (WO 2017/075531) and WO2020/146805, each of which is incorporated herein by reference in its entirety. Briefly, cationic lipid, DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of about 47.5:10:40.7:1.8.

Table 8. Lipids in lnp formulations

CAS = chemical abstracts service (ChemicalAbstract Service); DSPC = 1, 2-distearoyl-sn-glycero-3-phosphorylcholine

EXAMPLE 4 RSV preF expression

This example was used to capture in vitro expression (in vitro expression; IVE) results generated against Respiratory Syncytial Virus (RSV) modRNA Lipid Nanoparticle (LNP) drug products described herein. The constructs encapsulated in LNP encode full length RSV trimeric fusion glycoproteins (F-proteins) from strain a and B viruses, respectively.

TABLE 9 list of antibodies

Project	Source(s)	Concentration in analysis
			Human mAb anti-RSV mAb1	Inside (In-house)	500ng/mL
Mouse mAb against RSV L4-6	Inside part	500ng/mL
			Goat anti-mouse IgG AF647	Invitrogen	500ng/mL
Goat anti-human kappa-PE	Southern Biotech	500ng/mL

TABLE 10 list of test articles

Project	Test article
		1	LNP containing RSV F modRNA _847A
2	LNP containing RSV F modRNA _847B
		3	LNP containing RSV F modRNA _847A-fold
4	LNP with RSV F modRNA _851A
		5	LNP with RSV F modRNA _852A
6	LNP containing RSV F modRNA _DS-Cav1A
		7	LNP containing RSV F modRNA _wild type (WT) A
8	LNP containing RSV F SARNA _847A
		9	LNP containing RSV F SARNA _847B

The following protocol is described for the full staining (surface + intracellular protein), with the fixation and wash buffers changed to remove permeabilizing agents, all other steps and reagents being the same for the surface-only staining protocol. Briefly, 96-well culture plates were seeded with HEK293F cells at a density of 2.5×10 ⁵ cells/well and placed in a shaker incubator (350 rpm,37 ℃, wet, 5% CO ₂) while preparing for sample titration. LNP drug products were diluted in DPBS to a concentration of 80 ng/. Mu.L and serially diluted at 8 spots with a dilution factor of 4. Next, the 96-well culture dish was removed from the incubator and 50. Mu.L of diluted LNP for each step was added to the duplicate wells of the 96-well culture dish to generate a titration curve in the range of 8,000 ng/well to 1.95 ng/well. The 96-well culture dish was returned to the shake incubator overnight. After incubation, 250 μl of cells were transferred to 96-well u-bottom polystyrene culture plates and pooled (pelleted) using a swing bucket centrifuge (500 rcf,5min at room temperature). The supernatant was removed and the cells were resuspended in 100 μl Aqua405 live/dead staining solution. Incubate the plates at room temperature for 30min in the dark. After incubation, cells were washed with wash buffer and pelleted using centrifugation (500 rcf,5min at room temperature). The supernatant was removed and the cells were resuspended in 100 μl of fixation/permeation buffer and the culture dish incubated at 2-8 ℃ for 30min in the dark. Once incubation was complete, cells were pelleted using a swinging bucket centrifuge (500 rcf,5min at room temperature). The supernatant was removed and the cells resuspended with 250 μl wash buffer, which was repeated a total of 2 washes. After the final washing step, the cells were pelleted, the supernatant removed, and resuspended in 50 μl of primary antibody solution. The culture dish was sealed and incubated at 2-8℃for 45 minutes in the absence of light. Once completed, the cells were pelleted using a swinging bucket centrifuge (500 rcf,5min at room temperature). The supernatant was removed and the cells resuspended with 250 μl wash buffer, which was repeated a total of 2 washes. After the final washing step, the cells were pelleted, the supernatant removed, and resuspended in 50 μl of secondary antibody solution. The culture dish was sealed and incubated at 2-8℃for 45 minutes in the absence of light. Once completed, the cells were pelleted using a swinging bucket centrifuge (500 rcf,5min at room temperature). The supernatant was removed and the cells resuspended with 250 μl wash buffer, which was repeated a total of 2 washes. After the final washing step, the cells were pelleted, the supernatant removed, and resuspended in 200 μl wash buffer, and the data acquired by flow cytometry.

In Vitro Expression (IVE) of modRNA LNP pharmaceutical products was assessed by transfecting HEK293F cells, using dose titration curves, and performing antibody staining, namely RSV mAb1 specific for trimeric RSV F-protein and L4-6 specific for total RSV F-protein. These antibodies have been shown to recognize both strain a and strain B RSV F-proteins and are used in assays with osmotic or non-osmotic conditions to assess the total cell content and cell surface content of RSV F-proteins. The EC50 of the dose response curve of the measured positive cells% (2,000 ng/well input) and drug product lot are shown in table 11.

TABLE 11 IVE results for pharmaceutical products

Example 5 immune response (in vivo experiment)

Female BALB/c mice were vaccinated with the pre-RSV fusion F (847) in the form of a bivalent protein subunit (RSV 847a+847b) described in WO2017/109629 or the modRNA-LNP formulation in the form of a monovalent (RSV 847A) or bivalent (RSV 847a+847b) or saRNA-LNP in the form of a bivalent formulation described herein at different doses on day 0 and day 21. Immunogenicity was assessed by measuring RSV neutralizing antibody responses and RSV F specific T cell responses. Serum was collected for RSV neutralization analysis on day 21 and day 35 (2 weeks after dose 2 (PD 2)), and spleen was collected for T cell analysis (ELISpot and intracellular cytokine staining ICS analysis) on day 35 (2 2 weeks PD).

[ Neutralization analysis ]

RSV micro-neutralization assay is a 3-day assay using a549 cells (human alveolar basal epithelial cells) for measuring serum-neutralizing RSV activity as a functional antibody to prevent host cell monolayer infection. A549 cells (human alveolar basal epithelial cells; ATCC, cat. No. CCL-185) were seeded at 2.5×10 ⁴ cells/well on day 0 in 96-well tissue culture-treated culture plates and incubated for at least 20 hours to form confluent monolayers. On day 1, diluted virus (RSV a, M37; RSV B, B18537;500 FFU/well) was added to 3-fold serial dilutions of prepared heat-inactivated test serum, repeated twice, and incubated for 1 hour to allow antibodies to bind to the virus. Next, the neutralization reaction was transferred onto the prepared a549 cell monolayer and incubated for 2 hours. Additional medium was supplemented onto the culture dish prior to overnight incubation (at least 16 hours). On day 2, the culture dish was fixed with methanol and stained with a mouse primary antibody against RSV F (Pfizer, N50-9) followed by staining with a secondary antibody fluorescently labeled with Alexa 488 to detect viral foci. The 50% neutralization titer was calculated as the last serum dilution at which 50% of the virus was neutralized compared to wells containing virus alone. Titers are reported as Geometric Mean Titers (GMT) of two replicates of each sample. The analytical titer range is 20-43,740. Any samples with titers >43,740 were pre-diluted and repeated to expand the titer limit. Any sample below the lower limit of detection (LLOD) was reported at LLOD of 20.

[ T cell response measurement ]

Vaccine-induced T cell responses against RSV F were assessed by ex vivo stimulation of spleen cells in the presence of a pool of RSV F (a+b) peptides to activate the production of various cytokines, such as IFN- γ, in antigen-specific T cells. Cytokines secreted outside the cells can be measured by ELISpot (expressed as spot forming cells SFC/million cells), or cytokine secretion inside the cells measured by ICS (expressed as a percentage of CD4 ⁺ T cells and CD8 ⁺ T cells expressing cytokines) can be blocked.

In the case of ELISpot, the cytokine IFN- γ secreted by activated T cells is captured by anti-IFN- γ antibodies coated onto polyvinylidene fluoride (PVDF) membrane at the bottom of wells on the microplate. The captured IFN-gamma is developed as spots by another non-competitive biotin-labeled anti-IFN-gamma secondary antibody, followed by an enzymatic color reaction using streptococcal avidin-alkaline phosphatase (ALP) conjugate and a substrate solution of nitroblue tetrazolium and 5-bromo-4-chloro-3' -indolyl phosphate (BCIP/NBT-plus), which produces a dark purple precipitate or spot. T cell IFN-gamma responses were measured using the Mabtech mouse IFN-gamma ELISPot PLUS kit (ALP) and expressed as Spot Forming Cells (SFC)/million cells.

ICS staining can detect a variety of cytokines, including IFN- γ, produced in CD4 ⁺ and CD8 ⁺ T cells following antigenic peptide stimulation. In crpli with medium-DMSO (unstimulated) or specific peptide pools (15 aa,11aa overlap, 2 μg/mL/peptide) representing RSV fa+b, single cell suspensions of splenocytes (2×10 ⁶ cells/well) were cultured ex vivo for 5 hours at 37 ℃ in the presence of anti-CD 107a APC antibodies and protein transport inhibitors GolgiPlug and GolgiStop. After stimulation, spleen cells were incubated with fluorescent binding antibodies to surface proteins CD19, CD3, CD4, CD8, CD44 (25+ -5 min at 18-25 ℃) followed by fixation and permeation and staining for IFN-gamma, TNF-alpha, IL-2 and CD40L/CD154 (25+ -5 min at 18-25 ℃). After staining, cells were washed and resuspended in FC buffer. Cells were collected on LSR Fortessa and data analyzed by FlowJo (10.7.1). The results were background-subtracted (medium-DMSO) and shown as percentages of cytokine-expressing CD4 ⁺ T cells and CD8 ⁺ T cells.

[ As a result ]

[ In Vitro Expression (IVE) ]

The results of the IVE analysis of RNA-LNP are detailed in Table 11. In the modRNA-LNP formulation designed for RSV F, the trimeric configuration specific RSV mAb1, 847A-folder, DS-Cav1 and WT showed relatively lower expression, but the total F expression (L4-6) showed similar levels. Furthermore, the saRNA-LNP formulations of RSV 847A and 847B showed similar better expression for trimeric F and total F expression compared to modRNA-LNP. These results indicate that the partially stabilized full-length DS-Cav1 encoded from mRNA and native full-length WT F antigen present reduced proportions of antigen in the pre-fusion conformation compared to the full-length 847, 851 and 852 antigens from either RSV subgroup A or B. The limited pre-fusion mAb binding observed in the case of the soluble form of 847 (i.e., 847-foldlon) was due to proteins largely present in the cell culture supernatant following transient transfection, and the IVE method examined proteins that were either intracellular or localized only to the cell surface.

[ Immunogenicity ]

The results of neutralization assays on day 35 (week 2 PD 2) revealed that immunization of mice with modRNA-LNP formulations of RSV847 in monovalent or bivalent forms of RSV847A and 847B induced potent neutralizing antibody responses against both RSV a and RSV B that were higher compared to the bivalent protein subunit forms (fig. 1A and 1B, table 10). Furthermore, the RSV B neutralization response induced by the bivalent formulation RSV847a+rsv847b of modRNA-LNP was higher than that of the monovalent formulation RSV847A (fig. 1B, table 12), supporting the need for the bivalent form of RSV preFmodRNA vaccine for optimal immune response in humans.

The T cell response induced by these RSV preF modRNA-LNP formulations in mice was next assessed. IFN-. Gamma.ELISpot results showed that the RSV 847modRNA vaccine induced a potent T cell response with a trend similar to that of RSV neutralization (FIG. 1C, table 12). Furthermore, the ICS analysis results revealed that the RSV 847modRNA vaccine induced high frequency F-specific IFN-gamma expressing CD4 ⁺ T cells as well as CD8 ⁺ T cells in a dose-dependent manner (fig. 1D and 1E, table 12). Notably, the RSV a+bf-specific T cell response induced by the bivalent formulation (RSV 847a+847b) was higher than the monovalent formulation (RSV 847A), further supporting that the bivalent form of RSV 847 induced a higher magnitude of T cell response, similar to the neutralization response.

In summary, the results of the study demonstrate that the modRNA-LNP formulation of the F construct before RSV fusion is more immunogenic than the protein subunit vaccine in untreated mice.

TABLE 12 immune response in mice induced by the modRNA-LNP formulation of F before fusion of protein subunits and RSV

NA-unanalyzed

Next, the immunogenicity of modRNA-LNP encoding different RSV pre-fusion F (preF) antigen designs described herein was assessed. Neutralization assay results revealed that mutant pre-fusion designs 847, 851 and 852 in full length form induced higher titers than DS-CAV1 and WT and 847A-fold designed as ectodomain with fold (fig. 2). These results further confirm that the pre-fusion that stabilizes the mutation as well as the full length (ectodomain+tm+ct) form of the RSV F antigen is critical for optimal immunogenicity of RSV preF antigens when encoded from mRNA.

Self-amplifying RNA (saRNA) is an mRNA platform that utilizes RNA replicase to amplify mRNA encoding an antigen and is expected to be more potent than the modRNA platform. The immunogenicity of the saRNA-LNP was compared to modRNA-LNP and protein subunits using the divalent (a and B) formulation of RSVpreF. At 3 weeks post dose 1 (3 w PD1), dose-dependent effects were observed in neutralization reactions induced by modRNA and saRNA vaccines against RSV a and B viruses. Furthermore, the neutralization response induced by saRNA at the 0.02 μg dose was substantially higher than modRNA vaccine in mice receiving the 0.2 μg dose (fig. 3A and 3B), indicating a dose-saving response induced by saRNA. A strong dose-dependent neutralization response was induced by saRNA 2 weeks after dose 2 (2 w PD2), and was comparable to modRNA at a dose level of 0.2 μg for RSV a and B (fig. 3C and 3D). T cell responses in spleen analyzed by ICS analysis at 2w PD2 showed that robust and dose-dependent RSV a+ B F specific IFN-gamma expressing CD4 ⁺ and CD8 ⁺ T cell responses were induced by modRNA and saRNA vaccines (fig. 3E and 3F). The CD8 ⁺ T cell response induced by the saRNA group at the 0.02 μg dose level was substantially higher than the modRNA vaccine group at the 0.2 μg dose (fig. 3E and 3F), indicating a dose-sparing response induced by saRNA.

Taken together, these overall in vitro expression and immunogenicity results demonstrate that modRNA-LNP and saRNA-LNP, which encode RSV preF designs, support robust expression of RSV preF antigen in mice and produce efficient RSV F-specific immune responses.

EXAMPLE 6 RSV antigen

The sequences of the RSV antigens/polypeptides, RSV DNA and RSV RNA of the invention are provided in tables 1-3. The sequence may comprise any stop codon, including but not limited to the stop codons provided in the following table.

[ Table 1.RSV Polypeptides ]

[ Table 2.RSV DNA ]

[ Table 3.RSV RNA ]

The following paragraphs describe additional embodiments of the present invention:

1. an RNA molecule comprising at least one open reading frame encoding a Respiratory Syncytial Virus (RSV) polypeptide.

2. The RNA molecule of paragraph 1 wherein the RSV polypeptide is an RSV glycoprotein.

3. The RNA molecule of paragraph 2 wherein the RSV glycoprotein is RSV F protein.

4. The RNA molecule of any of paragraphs 1-3, wherein the RSV polypeptide is a full-length polypeptide, a truncated polypeptide, a fragment or variant thereof.

5. The RNA molecule of any of paragraphs 1-4, wherein the RSV polypeptide comprises at least one mutation.

6. The RNA molecule according to any of paragraphs 1-5, wherein the RSV polypeptide comprises the amino acids of Table 1, including but not limited to any of SEQ ID NOs 1-6 or 71-74.

7. The RNA molecule of any of paragraphs 1-6, wherein the RSV polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of any of SEQ ID NOs 1-6 or 71-74.

8. The RNA molecule according to any one of paragraphs 1-7, wherein the RSV polypeptide comprises the amino acid sequence of any one of SEQ ID NOS 1-6 or 71-74.

9. An RNA molecule according to any of paragraphs 1-8, wherein the open reading frame is transcribed from the nucleic acid sequence of Table 2, including but not limited to any of SEQ ID NOS 7-10 or 59-62.

10. An RNA molecule according to any one of paragraphs 1-9, wherein the open reading frame is transcribed from a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of any one of SEQ ID NOS: 7-10 or 59-62.

11. An RNA molecule according to any of paragraphs 1-10, wherein the open reading frame comprises the nucleic acid sequence of Table 3, including but not limited to any of SEQ ID NOS 11-16 or 63-70.

12. The RNA molecule according to any one of paragraphs 1-11, wherein the open reading frame comprises a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to the sequence of any one of SEQ ID NOS: 11-16 or 63-70.

13. An RNA molecule according to any one of paragraphs 1-12, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO 11-16 or 63-70.

14. The RNA molecule according to any one of paragraphs 1-13, wherein each uridine is replaced with N1-methyl pseudouridine (ψ).

15. The RNA molecule according to any one of paragraphs 1-14, further comprising a 5 'untranslated region (5' UTR).

16. An RNA molecule according to paragraph 15, wherein the 5' UTR comprises a sequence selected from any one of SEQ ID NOs 17 to 19.

17. The RNA molecule according to any one of paragraphs 1-16, further comprising a 3 'untranslated region (3' UTR).

18. A composition according to paragraph 17, wherein the 3' UTR comprises a sequence selected from any one of SEQ ID NOS: 20 to 25.

19. An RNA molecule according to any one of paragraphs 1-18, wherein the RNA molecule comprises a 5' cap moiety.

20. The RNA molecule according to any one of paragraphs 1-19, further comprising a 3' poly-A tail.

21. An RNA molecule according to paragraph 20, wherein the poly-A tail comprises a sequence having SEQ ID NO. 26, which comprises.+ -.1 or.+ -.2 adenosine (A) as appropriate.

22. The RNA molecule of any of paragraphs 1-21, wherein the RNA molecule comprises a 5'utr and a 3' utr.

23. The RNA molecule of any of paragraphs 1-22, wherein the RNA molecule comprises a 5' cap, a 5' utr, and a 3' utr.

24. The RNA molecule of any of paragraphs 1-23, wherein the RNA molecule comprises a 5' cap, a 5' utr, a 3' utr, and a poly-a tail.

25. An RNA molecule according to any one of paragraphs 1-24 comprising a 5'UTR comprising the sequence of SEQ ID NO:18 or 19, an open reading frame comprising the sequence of any one of SEQ ID NO: 11-16 or 63-70, and a 3' UTR comprising the sequence of SEQ ID NO: 20-25.

26. The RNA molecule according to any one of paragraphs 1-25 comprising a 5'UTR comprising the sequence of any one of SEQ ID NOS: 18 or 19, an open reading frame comprising the sequence of any one of SEQ ID NOS: 11-16 or 63-70, a 3' UTR comprising the sequence of any one of SEQ ID NOS: 20-25, and a poly A tail comprising the sequence of any one of SEQ ID NOS: 26.

27. An RNA molecule according to any of paragraphs 1-26, wherein the open reading frame is generated from codon optimized DNA.

28. The RNA molecule of any of paragraphs 1-27, wherein the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70% or at least 75%, or about 50% to 75% or 55% to 70%, or about 58%, 66% or 62%.

29. An RNA molecule according to any of paragraphs 1-28, wherein the encoded RSV polypeptide is located in the cell membrane, in the golgi apparatus and/or anchored in the membrane and secreted.

30. An RNA molecule according to any one of paragraphs 1-29, wherein the RNA molecule comprises stabilized RNA.

31. An RNA molecule according to any one of paragraphs 1-30, wherein the RNA comprises at least one modified nucleotide.

32. An RNA molecule according to paragraph 31, wherein the modified nucleotide is pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4 '-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydro-pseudouridine, 2-thio-dihydro-uridine, 2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 5-aza-uridine, dihydro-pseudouridine, 5-methoxy-uridine or 2' -O-methyl uridine.

33. The RNA molecule of paragraph 32 wherein the modified nucleotide is N1-methyl pseudouridine (ψ).

34. The RNA molecule according to any one of paragraphs 1-33, wherein the RNA is mRNA.

35. An RNA molecule according to paragraph 34, wherein the RNA is modRNA or saRNA.

36. A composition comprising the RNA molecule of any one of paragraphs 1-35, wherein the RNA molecule is formulated in a lipid nanoparticle (RNA-LNP).

37. A composition according to paragraph 36, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEG-lipid, a neutral lipid, and a steroid or steroid analog.

38. A composition according to paragraph 36 or 37, wherein the lipid nanoparticle comprises a cationic lipid.

39. A composition according to paragraph 38, wherein the cationic lipid is (4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyl decanoate) (ALC-0315).

40. The composition according to any one of paragraphs 36-39, wherein the lipid nanoparticle comprises a pegylated lipid.

41. A composition according to paragraph 40, wherein the pegylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC or PEG-CerC), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2- [ (polyethylene glycol) -2000] -N, N-ditetradecyl) acetamide, glycol-lipid (including PEG-c-DOMG, PEG-c-DMA, PEG-S-DMG, N- [ (methoxypolyethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoyloxy propyl-3-amine (PEG-c-DMA) and PEG-2000-DMG), pegylated diacylglycerol (PEG-DAG) (such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG)), pegylated phosphatidylethanolamine (PEG-PE), succinylated diacylglycerol (PEG-S-G) (such as 4-O- (2 ',3' -ditetradecyloxy) propyl-1-methoxy-O-D-co-PEG-4-O- ((PEG-ethyl) ceramide) Or PEG dialkoxypropyl carbamates such as co-methoxy (polyethoxy) ethyl-N- (2, 3- (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl-N- (ω -methoxy (polyethoxy) ethyl) carbamate.

42. A composition according to paragraph 40 or 41, wherein the PEG-lipid is 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159).

43. The composition according to any one of paragraphs 36-42, wherein the lipid nanoparticle comprises a neutral lipid.

44. A composition according to paragraph 43, wherein the neutral lipid is distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE) or 1, 2-dioleoyl-sn-glycerophosphate-3-trans PE.

45. A composition according to paragraph 43 or 44, wherein said neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC).

46. A composition according to any of paragraphs 36-45, wherein the lipid nanoparticle comprises a steroid or steroid analogue.

47. A composition according to paragraph 46, wherein the steroid or steroid analogue is cholesterol.

48. The composition according to any of paragraphs 36-47, wherein the lipid nanoparticle has an average diameter of about 1 to about 500nm.

49. The composition according to any one of paragraphs 36-48, wherein said RNA is from RSV subtype A.

50. The composition according to any one of paragraphs 36-48, wherein said RNA is from RSV subtype B.

51. The composition according to any one of paragraphs 36-48, wherein the RNA is derived from (i) RSV subtype A and (ii) RSV subtype B.

52. A composition according to any of paragraphs 36-51 comprising RNA molecules formulated in Lipid Nanoparticles (LNPs) at a concentration of or about 0.01-0.09 mg/mL, the lipid nanoparticles comprising cationic lipids at a concentration of or about 0.8-0.95 mg/mL, pegylated lipids at a concentration of or about 0.05-0.15 mg/mL, neutral lipids at a concentration of or about 0.1-0.25 mg/mL, and a steroid or steroid analogue at a concentration of or about 0.3-0.45 mg/mL.

53. A composition according to any of paragraphs 36-52 comprising RNA molecules formulated in Lipid Nanoparticles (LNP) at a concentration of or about 0.01-0.09 mg/mL, the lipid nanoparticles comprising (4-hydroxybutyl) aminoidene) bis (hexane-6, 1-diyl) bis (2-hexyldecanoate) (ALC-0315) at a concentration of or about 0.8-0.95 mg/mL, 2- [ (polyethylene glycol) -2000] -N, N-di (tetradecyl) acetamide (ALC-0159) at a concentration of or about 0.05-0.15 mg/mL, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC) at a concentration of or about 0.1-0.25 mg/mL, and cholesterol at a concentration of or about 0.3-0.45 mg/mL.

54. A composition according to any of paragraphs 36-53 comprising RNA molecules formulated in Lipid Nanoparticles (LNP) at a concentration of at or about 0.06 mg/mL.

55. A composition according to any of paragraphs 36-54, further comprising at least one of a buffer, a stabilizer, a salt, a surfactant, a preservative, an excipient and/or an adjuvant.

56. A composition according to any of paragraphs 36-55 further comprising at least a buffer and a stabilizer and optionally a salt diluent.

57. A composition according to paragraph 36 or 56, wherein the buffer is Tris buffer.

58. A composition according to paragraph 57, wherein the Tris buffer comprises tromethamine and Tris hydrochloric acid (HCl).

59. A composition according to paragraph 58, wherein the concentration of said bradykinin is at or about 0.1 to 0.3mg/mL or at or about 0.01 to 0.15mg/mL.

60. The composition of paragraphs 57 or 58, wherein the Tris HCl concentration is or about 1.25 to 1.40mg/mL or is or about 0.5 to 0.65mg/mL.

61. The composition according to any one of paragraphs 56-60, wherein the stabilizer is sucrose.

62. A composition according to paragraph 61, wherein the sucrose is at a concentration of or about 95 to 110mg/mL or at or about 35 to 50mg/mL.

63. The composition according to any of paragraphs 55-62, wherein the salt diluent for reconstitution is sodium chloride.

64. A composition according to paragraph 63, wherein the concentration of sodium chloride is at or about 5 to 15mg/mL.

65. A composition according to any of paragraphs 36-64, wherein the composition is liquid or lyophilized.

66. A composition according to paragraph 65 comprising RNA molecules formulated in Lipid Nanoparticles (LNP) at a concentration of or about 0.01-0.09 mg/mL, the lipid nanoparticles comprising cationic lipid at a concentration of or about 0.8-0.95 mg/mL, pegylated lipid at a concentration of or about 0.05-0.15 mg/mL, neutral lipid at a concentration of or about 0.1-0.25 mg/mL, and a steroid or steroid analogue at a concentration of or about 0.3-0.45 mg/mL, and further comprising Tris buffer comprising bradykinin at a concentration of or about 0.1-0.3 mg/mL, and Tris hydrochloric acid (HCl) at a concentration of or about 1.25-1.40 mg/mL, and sucrose at a concentration of or about 95-110 mg/mL, wherein the composition is a liquid composition.

67. A composition according to paragraph 65 comprising RNA molecules formulated in Lipid Nanoparticles (LNP) at a concentration of or about 0.01-0.09 mg/mL, the lipid nanoparticles comprising cationic lipid at a concentration of or about 0.8-0.95 mg/mL, pegylated lipid at a concentration of or about 0.05-0.15 mg/mL, neutral lipid at a concentration of or about 0.1-0.25 mg/mL, and a steroid or steroid analogue at a concentration of or about 0.3-0.45 mg/mL, and further comprising Tris buffer comprising bradykinin at a concentration of or about 0.01-0.15 mg/mL, and Tris HCl at a concentration of or about 0.5-0.65 mg/mL, sucrose at a concentration of or about 35-50 mg/mL.

68. A composition according to paragraph 65, wherein the composition is reconstituted with sodium chloride at a concentration of or about 5-15 mg/mL.

69. A composition according to paragraph 65, wherein the composition is reconstituted with or with about 0.6 to 0.75mg/mL sodium chloride.

70. A composition according to paragraph 65, further comprising or consisting of about 5 to 15mM Tris buffer, 200 to 400mM sucrose at pH or about 7.0 to 8.0, and optionally 0.9% sodium chloride diluent for reconstitution.

71. A method of inducing an immune response in an individual comprising administering to the individual an effective amount of an RNA molecule, RNA-LNP and/or composition of any of paragraphs 1-70.

72. A method of preventing, treating or ameliorating an infection, disease or condition in a subject comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or composition of any of paragraphs 1-70.

73. The method of paragraph 72, wherein the infection, disease or condition is associated with RSV.

74. A method according to paragraph 72 or 73, wherein the infection, disease or condition is an acute respiratory disorder induced by RSV infection, including pneumonia and bronchitis.

75. Use of the RNA molecule, RNA-LNP and/or composition of any of paragraphs 1-70 for the manufacture of a medicament for inducing an immune response against RSV in an individual.

76. Use of an RNA molecule, RNA-LNP and/or composition of any of paragraphs 1-70 for the manufacture of a medicament for preventing, treating or ameliorating an infection, disease or condition in a subject.

77. The use according to paragraph 76, wherein the infection, disease or condition is associated with RSV.

78. The use according to paragraph 76 or 77, wherein the infection, disease or condition is a respiratory disorder associated with RSV infection, including pneumonia and bronchitis.

79. A method or use according to any of paragraphs 71-78, wherein the individual is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

80. A method or use according to any of paragraphs 71-79, wherein the individual is about 50 years old or older.

81. The method or use according to any one of paragraphs 71-79, wherein the subject is a pregnant woman.

82. The method or use according to any one of paragraphs 71-81, wherein the RNA molecule or composition is administered as a vaccine.

83. The method or use according to any of paragraphs 71-82, wherein the RNA molecule or composition is administered by intradermal or intramuscular injection.

84. An RNA molecule according to paragraph 83, wherein the modRNA comprises the nucleotide having SEQ ID NO. 13.

85. An RNA molecule according to paragraph 83, wherein the modRNA comprises the nucleotide having SEQ ID NO. 14.

86. An RNA molecule according to paragraph 83, wherein the sarNA comprises the nucleotide with SEQ ID NO. 15.

87. An RNA molecule according to paragraph 83, wherein the modRNA comprises the nucleotide having SEQ ID NO. 16.

88. The method or use according to any of paragraphs 71-87, wherein a single dose, two doses, three doses or more of said RNA molecule and/or composition are administered to the subject.

89. A method or use according to any of paragraphs 71-88, wherein a single dose of said RNA molecule and/or composition is administered to the subject.

90. A method or use according to any of paragraphs 71-89, wherein said RNA molecule and/or composition is administered to said subject in two doses.

91. A method or use according to any of paragraphs 71-90, wherein the subject is administered two doses of said RNA molecule and/or composition on day 0 and after or about 2 months.

92. A method or use according to any of paragraphs 71-91, wherein the subject is administered two doses of said RNA molecule and/or composition on day 0 and after or about 6 months.

93. A method or use according to any of paragraphs 71-92, wherein at least one booster dose of said RNA molecule and/or composition is administered to the subject.

94. A method or use according to any of paragraphs 71-93, wherein each administration administers at least or at least about 15 μg, 30 μg, 60 μg, 90 μg, 100 μg or higher dose of RNA molecule and/or composition to the subject.

95. The method or use according to any of paragraphs 71-94, wherein the volume of injection solution is administered to the subject at or about 0.25-1 mL, including but not limited to at or about 0.25, 0.5, 1 mL.

All methods disclosed and claimed herein can be performed and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of certain embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. Rather, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

All cited references recited in this application (including literature references, issued patents, published patent applications, and published patent applications as cited throughout this applicationAccession number) are specifically and explicitly incorporated herein by reference to the extent that they provide exemplary procedures or other details that complement those set forth herein. To the extent that the definition of a term in a document, which is incorporated by reference herein conflicts with the definition used herein, the definition used herein controls.

Claims

1. An RNA molecule comprising at least one open reading frame encoding a respiratory syncytial virus (RSV) fusion protein F (F) polypeptide.

2. An RNA molecule according to claim 1, wherein the RSV polypeptide is a full-length polypeptide, a truncated polypeptide, a fragment or a variant thereof.

3. An RNA molecule according to claim 1, wherein the RSV polypeptide comprises at least one mutation.

4. The RNA molecule according to claim 1, wherein the RSV polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from SEQ ID NOs: 1-6 and 71-74.

5. The RNA molecule according to claim 1, wherein the open reading frame is transcribed from a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the sequences of SEQ ID NOs: 7-10 and 59-62.

6. The RNA molecule according to claim 1, wherein the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the sequences of SEQ ID NOs: 11 to 16 and 63 to 70.

7. The RNA molecule according to claim 1, wherein the open reading frame comprises the nucleic acid sequence of any one of SEQ ID NOs: 11 to 16 and 63 to 70.

8. The RNA molecule according to claim 1, further comprising a 5' untranslated region (5'UTR).

9. The RNA molecule according to claim 8, wherein the 5'UTR comprises a sequence selected from any one of SEQ ID NOs: 17 to 19.

10. The RNA molecule according to claim 1, further comprising a 3' untranslated region (3'UTR).

11. The RNA molecule according to claim 10, wherein the 3'UTR comprises the sequence of any one of SEQ ID NOs: 20 to 25.

12. The RNA molecule according to claim 1, wherein the RNA molecule further comprises a 5' cap portion or a 3' poly A tail.

13. The RNA molecule according to claim 12, wherein the poly A tail comprises a sequence having SEQ ID NO: 26.

14. The RNA molecule according to claim 1, wherein the open reading frame comprises a G/C content of at least 55%, 60%, 65%, 70% or 75%, or at or about 50% to 75% or 55% to 70%.

15. The RNA molecule according to claim 1, wherein the encoded RSV polypeptide is localized in the cell membrane, localized in the Golgi apparatus and/or is secreted.

16. The RNA molecule according to claim 1, wherein the RNA comprises at least one modified nucleotide.

17. The RNA molecule according to claim 16, wherein the modified nucleotide is pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine.

18. The RNA molecule according to claim 17, wherein the modified nucleotide is N1-methylpseudouridine (Ψ).

19. The RNA molecule according to claim 1, wherein the RNA is mRNA.

20. The RNA molecule according to claim 19, wherein the RNA is modRNA or saRNA.

21. A composition comprising an RNA molecule according to claim 1, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

22. The composition according to claim 21, wherein the lipid nanoparticles comprise at least one of: a cationic lipid, a pegylated lipid, a neutral lipid, and a steroid or a steroid analog.

23. The composition according to claim 22, wherein the cationic lipid is (4-hydroxybutyl)amino)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

24. The composition of claim 22, wherein the PEGylated lipid is PEG-modified phosphatidylethanolamine; PEG-modified phosphatidic acid; PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20); PEG-modified dialkylamine; PEG-modified diacylglycerol; PEG-modified dialkylglycerol; 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide; glycol lipids including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000)carbamoyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG; PEGylated diacylglycerol oil (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); PEGylated phosphatidylethanolamine (PEG-PE); PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); PEGylated ceramide (PEG-cer); or PEG dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

25. The composition according to claim 24, wherein the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159).

26. The composition according to claim 22, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-( N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

27. The composition according to claim 26, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

28. The composition according to claim 22, wherein the steroid or steroid analog is cholesterol.

29. A method of inducing an immune response against RSV in an individual, comprising administering to the individual an effective amount of an RNA molecule according to claim 1.

30. A method for preventing, treating or ameliorating an infection, disease or condition associated with RSV in an individual, comprising administering to the individual an effective amount of an RNA molecule according to claim 1.

31. The method according to claim 30, wherein the infection, disease or condition is an acute respiratory disorder induced by RSV infection, including pneumonia and bronchitis.

32. The method according to claim 29 or 30, wherein the individual is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

33. A method according to claim 29 or 30, wherein the RNA molecule is administered as a vaccine.

34. The method according to claim 29 or 30, wherein the subject is administered a single dose, two doses, three doses or more doses of the RNA molecule and optionally additional doses of the RNA molecule.

35. A method of inducing an immune response against RSV in an individual, comprising administering to the individual an effective amount of a composition according to claim 22.

36. A method for preventing, treating or ameliorating an infection, disease or condition associated with RSV in a subject, comprising administering to the subject an effective amount of a composition according to claim 22.