RNA molecule encoding RSV-F and vaccine containing same

Abstract

The present invention relates to RNA molecules encoding respiratory syncytial virus (RSV). The invention further relates to a composition comprising said RNA molecule (RNA-LNP) formulated in a lipid nanoparticle. 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 tract conditions, including pneumonia and bronchitis.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 585,254, filed on September 26, 2023, and U.S. Provisional Application No. 63 / 381,238, filed on October 27, 2022. The entire content of each of the foregoing applications is incorporated herein by reference.

[0003] Reference to Sequence Listing

[0004] This application is being filed via EFS - Web and includes a sequence listing submitted electronically in.xml format. The.xml file contains a sequence listing named "PC072895A Sequence Listing.xml", which was created on September 25, 2023, and is 145 KB in size. The sequence listing contained in this.xml file is part of the specification and is incorporated herein by reference in its entirety.

[0005] Background Art

[0006] Respiratory syncytial virus (RSV) is a respiratory virus that infects the lungs and respiratory tract. RSV is the leading cause of severe viral lower respiratory tract illness in infants worldwide and an important cause of respiratory illness in the elderly. In 2023, two RSV protein subunit vaccines were approved: ABRYSVO (Pfizer) and AREXVY (GSK). However, no RNA vaccines have been approved for the prevention of RSV infection.

[0007] RSV is a member of the family Pneumoviridae. Its genome consists of a single - stranded, antisense RNA molecule encoding 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two non - structural proteins. The structural proteins include three transmembrane surface glycoproteins: attachment protein G, fusion protein F, and small hydrophobic SH protein. There are two RSV subtypes: A and B. They differ mainly in the G glycoprotein, while the sequences of the F glycoprotein are more conserved between the two subtypes.

[0008] The mature F glycoprotein has three general domains: an extracellular domain (ED), a transmembrane domain (TM), and a cytoplasmic tail (CT). The CT contains a single palmitoylated cysteine residue.

[0009] The F glycoprotein of human RSV is initially translated from mRNA as a single 574 amino acid polypeptide precursor (referred to as "F0" or "F0 precursor"), which contains a signal peptide sequence (amino acids 1-25) at the N-terminus. After translation, the signal peptide is removed by signal peptidase in the endoplasmic reticulum. The remaining portion of the F0 precursor (i.e., residues 26-574) can be cleaved by cellular proteases (specifically furin) at two polybasic sites (a.a. 109 / 110 and 136 / 137), thereby removing a 27 amino acid insert sequence called pep27 (amino acids 110-136) and generating two linked fragments called 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 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. Either the uncleaved F0 protein without the signal peptide sequence or the F1-F2 heterodimer can form the RSV F protomer. Three such protomers assemble to form the final RSV F protein complex, which is a homotrimer of three protomers.

[0010] The F proteins of subtypes A and B are approximately 90% identical in amino acid sequence. An exemplary sequence of the F0 precursor polypeptide of subtype A is provided in SEQ ID NO: 1 (A2 strain; GenBank GI: 138251; Swiss Prot P03420), and an exemplary sequence of the F0 precursor polypeptide of subtype B is provided in SEQ ID NO: 2 (18537 strain; GenBank GI: 138250; Swiss Prot P13843). Both SEQ ID NO: 1 and SEQ ID NO: 2 are 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 1-25 (GenBank and UniProt). In both sequences, the TM domain is approximately amino acids 530-550, but alternatively has been reported as 525-548. The cytoplasmic tail begins at amino acid 548 or 550 and ends at amino acid 574, with a palmitoylated cysteine residue at amino acid 550.

[0011] The RSV F protein is the primary antigen for exploring RSV vaccines. The RSV F protein trimer mediates the fusion between the viral particle membrane and the host cell membrane and also promotes the formation of syncytia. In viral particles before membrane fusion with the host cell, the largest population of F molecules forms a lollipop-shaped structure, where the TM domain is anchored in the viral envelope [Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012.]. This conformation is called the pre-fusion conformation. Pre-fusion RSV F is recognized by monoclonal antibodies (mAbs) D25, AM22, and MPE8, with no difference among oligomeric states. The pre-fusion F trimer is 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 the pre-fusion state (which may be referred to herein as "pre-F") to the post-fusion state ("post-F") via an intermediate extended 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 engage three additional helices to form a post-fusion six-helix bundle. If the pre-fusion RSV F trimer is subjected to increasingly harsh chemical or physical conditions such as high temperature, it undergoes structural changes. Initially, the trimeric structure is lost (at least locally within the molecule) and then rearranged into the post-fusion form, and then domain denaturation occurs.

[0012] To prevent virus entry, F-specific neutralizing antibodies may have to bind to the pre-fusion conformation of F on the virus particle or a possible extended intermediate before virus envelope and cell membrane fusion. Thus, the pre-fusion form of the F protein is considered the preferred conformation as a desired vaccine antigen [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)]. The F glycoprotein readily converts to the post-fusion form upon extraction from membranes with surfactants (such as Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octyl thioglucoside, SDS, CHAPS, CHAPSO), or expression as an extracellular domain, physical or chemical stress, or after storage [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. January 20, 2010; 396(2):226-37]. Thus, preparation of pre-fusion F as a vaccine antigen remains a challenge.Due to neutralization and the protective antibody function of preventing virus entry, it is assumed that the F antigen that does not elicit pre-fusion specific antibodies is not expected to be as effective as the F antigen that elicits pre-fusion specific antibodies. Therefore, it is considered more necessary to utilize an F protein vaccine containing an F protein immunogen in a pre-fusion form. Mutants of the RSV F protein have been provided to increase pre-fusion stability (see, for example, PCT application No. WO2017 / 109629) and are promising vaccine candidates.

[0013] RSV vaccines incorporating F protein antigens are under development. Clinical studies have shown that some F protein subunit-based vaccine candidates are safe and immunogenic, but improvements are needed in terms of protective efficacy and duration of protection.

[0014] Therefore, there is a need for improved immunogenic compositions to protect against RSV infection. SUMMARY OF THE INVENTION

[0015] The present invention addresses the unmet need for improved immunogenic compositions against RSV infection, particularly as provided herein. In one embodiment, the present 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 (such as 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 an RSV protein for inducing an immune response against RSV in an individual. The immunogenic RNA polynucleotide encoding the immunogenic antigen is administered to provide an antigen for inducing (such as stimulating, initiating, and / or amplifying) an immune response (such as antibodies and / or immune effector cells) (after the polynucleotide is expressed by appropriate target cells). In one embodiment, the immune response induced according to the present invention is both a B cell-mediated immune response (such as an antibody-mediated immune response) and a T cell-mediated immune response. In one embodiment, the immune response is an anti-RSV immune response.

[0016] The immunogenic compositions described herein comprise RNA molecules that contain RNA (as the active ingredient) that can be translated into one or more proteins in the cells of a recipient. In addition to wild-type, codon-optimized, or mutant sequences encoding antigenic sequences, the RNA molecules can contain one or more structural components (5' cap, 5' UTR, subgenomic promoter, 3' UTR, polyA tail) optimized for maximal 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 (such as lipid nanoparticles (LNPs)) for administration. In one embodiment, the RNA molecules described herein are complexed with lipids to produce RNA-lipid nanoparticles (such as RNA-LNPs) for administration. In one embodiment, the RNA molecules described herein are complexed with proteins 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 can be complexed together with lipids and / or proteins or separately with lipids and / or proteins to produce RNA-particles for administration.

[0017] The present invention provides RNA molecules and RNA-LNPs that include at least one 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 the RSV F protein. In some embodiments, the RSV F protein is a full-length protein, a truncated protein, a fragment thereof, or a variant. In some embodiments, the RSV F protein contains at least one mutation.

[0018] The present invention provides RNA molecules and RNA-LNPs that include 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 polypeptide has, has at least, or has at most 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98%, or 99% or higher identity to any one of the amino acid sequences of Table 1 (such as any one of SEQ ID NOs: 1-6 or 71-74). In some embodiments, the RSV polypeptide consists of any one of the amino acid sequences of Table 1 (such as any one of SEQ ID NOs: 1-6 or 71-74).

[0019] The present invention provides an RNA molecule and an RNA-LNP, which comprise at least one open reading frame (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 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 having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher identity to any one of the nucleic acid sequences in Table 2 (e.g., any one 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 one of the nucleic acid sequences in Table 2 (e.g., any one of SEQ ID NOs: 7-10 or 59-62).

[0020] The present invention further provides an RNA molecule and an RNA-LNP, which comprise at least one ORF comprising an RNA nucleic acid sequence 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 one of the nucleic acid sequences in Table 3 (e.g., any one of SEQ ID NOs: 11-16 or 63-70). In some embodiments, the RNA molecule comprises a nucleic acid sequence consisting of any one of the nucleic acid sequences in Table 3 (e.g., any one of SEQ ID NOs: 11-16 or 63-70). In some embodiments, each uridine of any one of SEQ ID NOs: 11-16 is replaced with N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

[0021] The present invention further provides an RNA molecule and an RNA-LNP, which include a 5' untranslated region (5'-UTR) and / or a 3' 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: 17-19. In some embodiments, the 5'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher 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: 17-19. In some embodiments, the 5'UTR comprises a sequence consisting of any one of SEQ ID NOs: 17-19.

[0022] In some embodiments, the RNA molecule and the RNA-LNP 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 higher 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: 20-25.

[0023] The present invention further provides an RNA molecule and an RNA-LNP, which include a 5' cap portion. In some embodiments, the 5' cap portion is (3′OMe)-m 2 7,3'-O Gppp(m 1 2'-O )ApG. The present invention further provides an RNA molecule and an RNA-LNP, which include a 3' poly-A tail. In some embodiments, the poly-A tail comprises a sequence having SEQ ID NO: 26.

[0024] 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 molecule includes a 5′UTR, a 3′UTR and a poly-A tail. In some embodiments, one, two, three or more of the foregoing elements may be excluded from the RNA molecule. In some embodiments, each uridine of any one of the 5'UTR, 3′UTR and poly-A tail is replaced with N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

[0025] In some embodiments, the poly-A tail length may contain +1 / -1A. In some embodiments, the uridine is N1-methylpseudouridine (Ψ).

[0026] The present invention provides the 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 polyA 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 polyA 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 polyA 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 polyA 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 polyA 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 polyA tail of SEQ ID NO: 26. In some embodiments, the RSV ORF further comprises the stop codons described herein. In some embodiments, the polyA tail length may contain +1 / -1 A or +2 / -2 A. In some embodiments, each uridine of the RNA molecule is replaced with N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

[0027] The present invention further provides an RNA molecule, which comprises at least one open reading frame generated from codon-optimized DNA. In some embodiments, the open reading frame comprises at least, at most, exactly, or between any two of the following (inclusive or exclusive): a G / C content of 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%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, is or is about 50% - 75%, or is or is about 55% - 70%. In some embodiments, the G / C content is or is about 58%, is or is about 66%, or is or is about 62%.

[0028] The present invention further provides an RNA molecule, which comprises stabilized RNA. The present invention further provides an RNA molecule, which comprises an RNA having at least one modified nucleotide (e.g., modified RNA; modRNA). In some embodiments, the modified nucleotide is pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 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-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, or 2'-O-methyluridine. In some embodiments, the modified nucleotide is N1-methylpseudouridine (Ψ). In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing modified nucleotides may be excluded from the RNA molecule.

[0029] The present invention further provides an RNA molecule, which is messenger RNA (mRNA) or self-replicating RNA. In some embodiments, the RNA is mRNA.

[0030] The present invention further provides an immunogenic composition comprising the RNA molecules described herein. The RNA molecules can be formulated in, encapsulated in, complexed with, bound to, or adsorbed onto lipid nanoparticles (LNPs) in such immunogenic compositions (e.g., RSV RNA-LNP). In some embodiments, the lipid nanoparticles comprise at least one of the following: cationic lipids, polymer-conjugated lipids (e.g., polyethylene glycolylated lipids), and at least one structural lipid (e.g., neutral lipids and steroids or steroid analogs). In some embodiments, one, two, three, or more of the foregoing lipids can be excluded from the lipid nanoparticles.

[0031] In some embodiments, the lipid nanoparticles comprise cationic lipids. In some embodiments, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

[0032] In some embodiments, the lipid nanoparticles comprise polymer-conjugated lipids. In some embodiments, the lipid nanoparticles comprise polyethylene glycolated lipids, also known as PEG-lipids. In some embodiments, the polyethylene glycolated 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-bis(tetradecyl)acetamide; diol-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; polyethylene glycolated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); polyethylene glycolated 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); polyethylene glycolated ceramide (PEG-cer); or PEG dialkoxypropyl carbamate, 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 polyethylene glycolated lipids may be excluded from the RNA molecule. In some embodiments, the polyethylene glycolated lipid is 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

[0033] 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-phosphocholine (DSPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl 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), 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) and / or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing structural lipids may be excluded from the RNA molecule. In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0034] In some embodiments, the lipid nanoparticle comprises a second structural lipid, such as a steroid or a steroid analogue. In some embodiments, the steroid or steroid analogue is cholesterol.

[0035] In some embodiments, the average diameter of the lipid nanoparticle is about 1 to about 500 nm, such as at least, at most, exactly below or between any two of the following (inclusive or exclusive): 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm or 500 nm.

[0036] In some embodiments, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA molecule / polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably or about 0.01 - 0.09 mg / mL, encapsulated in an LNP composed of lipids comprising: a cationic lipid at a concentration of or about 0.8 - 0.95 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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 polyethylene glycolylated lipid at a concentration of or about 0.05 - 0.15 mg / mL (e.g., at least, at most, exactly 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 0.15 mg / mL), a first structural lipid at a concentration of or about 0.1 - 0.25 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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, or 0.25 mg / mL), and a second structural lipid at a concentration of or about 0.3 - 0.45 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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 buffering composition comprising a first buffer at a concentration of or about 0.1 to 0.3 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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), a second buffer at a concentration of or about 1.25 to 1.4 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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 a concentration of or about 95 to 110 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 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.

[0037] In certain embodiments, the liquid RNA-LNP immunogenic composition comprises an RNA molecule / polynucleotide encoding an RSV polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably or about 0.01 - 0.09 mg / mL, encapsulated in an LNP composed of lipids comprising: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of or about 0.8 - 0.95 mg / mL (e.g., at least, at most, exactly, or between any two of the following (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), 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159) at a concentration of or about 0.05 - 0.15 mg / mL (e.g., at least, at most, exactly, 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 0.15 mg / mL), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of or about 0.1 - 0.25 mg / mL (e.g., at least, at most, exactly, or between any two of the following (inclusive 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, or 0.25 mg / mL), and cholesterol at a concentration of or about 0.3 - 0.45 mg / mL (e.g., at least, at most, exactly, or between any two of the following (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 Tris buffer composition, the Tris buffer composition comprising tris(hydroxymethyl)aminomethane at a concentration of or about 0.1 to 0.3 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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 Tris hydrochloride (HCl) at a concentration of or about 1.25 to 1.4 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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 sucrose at a concentration of or about 95 to 110 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 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 concentrations of the foregoing elements may be excluded from the liquid RNA-LNP composition.

[0038] In some embodiments, the liquid RNA-LNP immunogenic composition comprises an RNA molecule / polynucleotide encoding an RSV polypeptide as disclosed herein at a concentration of at least, at most, exactly, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably or about 0.01 - 0.09 mg / mL, encapsulated in an LNP, and further comprises a Tris buffer at or about 5 - 15 mM (e.g., at least, at most, exactly, or between any two of the following (inclusive or exclusive): 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, at most, exactly, or between any two of the following (inclusive or exclusive): 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mM), with a pH of or about 7.0 - 8.0 (e.g., at least, at most, exactly, or between any two of the following (inclusive or exclusive): 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). In some embodiments, one, two, three, or more of the foregoing elements may be excluded from the liquid RNA-LNP composition. In some embodiments, one, two, three, four, five, or more of the foregoing element concentrations may be excluded from the liquid RNA-LNP composition.

[0039] In some embodiments, the RNA-LNP immunogenic composition is a lyophilized (reconstituted) RNA-LNP composition comprising an RNA molecule / polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably or about 0.01 - 0.09 mg / mL, encapsulated in an LNP composed of lipids comprising: a cationic lipid at a concentration of or about 0.8 - 0.95 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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 polyethylene glycolylated lipid at a concentration of or about 0.05 - 0.15 mg / mL (e.g., at least, at most, exactly 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 0.15 mg / mL), a first structural lipid at a concentration of or about 0.1 - 0.25 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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, or 0.25 mg / mL), and a second structural lipid at a concentration of or about 0.3 - 0.45 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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 each of the following: a first buffer at a concentration of or about 0.01 and 0.15 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive 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 second buffer at a concentration of or about 0.5 and 0.65 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive 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 stabilizer at a concentration of or about 35 - 50 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / mL), and a salt diluent for reconstitution at a concentration of or about 5 - 15 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL). In certain embodiments, the lyophilized composition is reconstituted in or with about 0.6 - 0.75 mL of salt diluent (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 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 concentrations in the lyophilized RNA-LNP composition are 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.

[0040] In certain embodiments, the lyophilized (reconstituted) RNA-LNP composition comprises an RNA polynucleotide encoding an RSV polypeptide herein disclosed at a concentration of at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg / mL, preferably or about 0.01 - 0.09 mg / mL, encapsulated in an LNP of a lipid composition comprising: ALC-0315 at a concentration of or about 0.8 - 0.95 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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), ALC-0159 at a concentration of or about 0.05 - 0.15 mg / mL (e.g., at least, at most, exactly 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 0.15 mg / mL), DSPC at a concentration of or about 0.1 - 0.25 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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, or 0.25 mg / mL), and cholesterol at a concentration of or about 0.3 - 0.45 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (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, the Tris buffer composition comprising: tromethamine at a concentration of or about 0.01 - 0.15 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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), and at a concentration of or about 0.5 - 0.65 mg / mL (e.g., at least, at most, exactly below, or between any two of the following (inclusive 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) of Tris HCl, a concentration of or about 35 - 50 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg / mL) of sucrose, and a concentration of or about 5 - 15 mg / mL (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg / mL) of sodium chloride (NaCl) diluent for reconstitution. In certain embodiments, the lyophilized composition is reconstituted in or with about 0.6 - 0.75 mL of sodium chloride (such as at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 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.

[0041] The present invention provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered to an individual at a dose of at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or higher of RSV RNA encapsulated in an LNP. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing concentrations of RSV RNA encapsulated in an LNP may be excluded.

[0042] The present invention provides RNA molecules, RNA-LNPs and immunogenic compositions, which can be administered in a single dose. The present invention further provides RNA molecules, RNA-LNPs and immunogenic compositions, which 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 1 month later, day 0 and at or about 2 months later, day 0 and at or about 3 months later, day 0 and at or about 6 months later, day 0 and at or about 9 months later, day 0 and at or about 12 months later, day 0 and at or about 18 months later, day 0 and at or about 2 years later, day 0 and at or about 5 years later, or day 0 and at or about 10 years later). The present invention further provides RNA molecules, RNA-LNPs and immunogenic compositions, which can be administered twice on day 0 and at or about 2 months later. The present invention further provides RNA molecules, RNA-LNPs and immunogenic compositions, which can be administered twice on day 0 and at or about 6 months later. The present invention further provides RNA molecules, RNA-LNPs and immunogenic compositions, which can be administered three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more. In some embodiments, periodic boosters at intervals of 1 to 5 years may be required to maintain the protective level of antibodies. The present invention further provides the administration of at least one booster dose. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing administration regimens may be excluded.

[0043] The present invention provides a method of inducing an immune response against RSV in an individual, which comprises administering to the individual an effective amount of the RNA molecules, RNA-LNPs and / or immunogenic compositions described herein. The present 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.

[0044] The present invention provides a method of inducing an immune response against RSV in an individual, which comprises 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 present invention further provides 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.

[0045] The present invention provides a method for inducing an immune response against RSV in an individual, which comprises administering to the individual an effective amount of an RNA molecule and / or an RNA-LNP, or a composition, as described herein, comprising at least one open reading frame encoding a polypeptide of interest. The present invention further provides the use of an RNA molecule and / or an RNA-LNP, or a composition, as described herein, comprising at least one open reading frame encoding a polypeptide of interest, for the manufacture of a medicament for inducing an immune response against RSV in an individual.

[0046] The present invention provides a method for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual, which comprises administering to the individual an effective amount of an RNA molecule, an RNA-LNP, and / or an immunogenic composition as described herein. The present invention further provides the use of an RNA molecule, an RNA-LNP, and / or an immunogenic composition as described herein, for the manufacture of a medicament for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual. In some embodiments, the infection, disease, or medical condition is related to RSV. In some embodiments, the infection, disease, or medical condition is acute lower respiratory infection (ALRI), including pneumonia and bronchitis. In some embodiments, the infection, disease, or medical condition is acute lower respiratory infection (ALRI), including pneumonia and bronchitis.

[0047] The present invention provides a method for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual, which comprises administering to the individual an effective amount of an RNA molecule and / or an RNA-LNP, or an immunogenic composition, as described herein, comprising at least one open reading frame encoding an RSV polypeptide. The present invention further provides the use of an RNA molecule and / or an RNA-LNP, or an immunogenic composition, as described herein, comprising at least one open reading frame encoding an RSV polypeptide, for the manufacture of a medicament for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual. In some embodiments, the infection, disease, or medical condition is related to RSV. In some embodiments, the infection, disease, or medical condition is acute lower respiratory infection (ALRI), including pneumonia and bronchitis. In some embodiments, the infection, disease, or medical condition is acute lower respiratory infection (ALRI), including pneumonia and bronchitis.

[0048] The present invention further provides a method for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual, comprising administering to the individual an effective amount of an RNA molecule and / or RNA-LNP comprising at least one open reading frame encoding a polypeptide of interest as described herein, or an immunogenic composition. The present invention further provides the use of an RNA molecule and / or RNA-LNP comprising at least one open reading frame encoding a polypeptide of interest as described herein, or an immunogenic composition, for the manufacture of a medicament for preventing, treating, and / or ameliorating an infection, disease, or medical condition in an individual. In some embodiments, the infection, disease, or medical condition is related to the gene of interest.

[0049] In some embodiments, the age of the individual is at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or the age is 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 age of the individual is, is at least, is at most, or is 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, or older. In some embodiments, the age of the individual is or is about 50 years old or older. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing age groups do not receive the RNA molecule and / or RNA-LNP.

[0050] In some embodiments, the individual has immunocompetence. In some embodiments, the individual is immunocompromised.

[0051] The present 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 present 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.

[0052] Upon consideration, any embodiment discussed in this specification can be implemented with respect to any method or composition of the present invention, and vice versa. Furthermore, the compositions of the present invention can be used to implement the methods of the present invention.

[0053] Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect can also be described in the "use" claim language, such as the "use" of any compound, composition, or medicament as described herein for achieving or implementing the described therapeutic, diagnostic, or physiological purpose or effect. The use of one or more compositions can be adopted based on any method described herein.

[0054] Other objects, features, and advantages of the present invention will become apparent from the following embodiments. However, it should be understood that the embodiments and specific examples, although indicating specific embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figures 1A to 1E Showing the immunogenicity of the modRNA-LNP formulation of RSV 847 in mice. On days 0 and 21, female BALB / c mice (10 per group) were immunized intramuscularly with RSV 847 constructs in the form of a bivalent protein subunit (RSV 847A + B) or in the form of a monovalent (RSV 847A) or bivalent (RSV 847A + B) modRNA-LNP formulation at the indicated doses. On day 35 (2 weeks after dose 2 (PD2)), sera were collected for RSV neutralization assays, and spleens were collected for T cell analysis (ELISpot and intracellular cytokine staining ICS assays). Figure 1A and Figure 1B Showing the results of the neutralization assay for RSV A and B, which are expressed as 50% neutralization titers (each symbol represents the titer from an individual animal. Bars represent the geometric mean titer (GMT)). Figure 1C Showing the results of the ELISpot assay, which measures the number of RSV A + B F-specific cells secreting IFN-γ and is expressed as spot-forming cells (SFC) / million cells. Figure 1D and Figure 1E Showing the results of the ICS assay, which measures the cells expressing RSV A + B F-specific IFN-γ within CD4 + and CD8 + T cells, expressed as the percentage of IFN-γ+ cells. Bars and error bars depict the median and interquartile range. NA: Not analyzed.

[0057] Figure 2 Showing the immunogenicity of the modRNA-LNP formulation encoding different RSV A prefusion F (preF) designs in mice. On days 0 and 21, female BALB / c mice (10 per group) were immunized intramuscularly with the modRNA-LNP formulation encoding the RSV A prefusion F (preF) designs described herein at a dose of 0.5 μg. On day 35 (2W PD2), the RSV A neutralization response of the sera was analyzed and expressed as 50% neutralization titers. Each symbol represents the titer from an individual animal. Bars represent the geometric mean titer (GMT).

[0058] Figures 3A to 3F Show the immunogenicity of modRNA-LNP and saRNA-LNP formulations of RSV prefusion F (preF) in mice. On days 0 and 21, female BALB / c mice (10 per group) were immunized intramuscularly at the indicated doses with RSV preF constructs in the form of a bivalent protein subunit (RSV preF A+B) or a bivalent modRNA-LNP formulation or a bivalent saRNA-LNP formulation. On days 21 (3W PD1) and 35 (2W PD2), sera were collected for RSV neutralization assays, and spleens were collected on day 35 for T cell analysis (intracellular cytokine staining ICS assay). Show the results of the neutralization assays for RSV A and B, expressed as the 50% neutralization titers at 3W PD1 ( Figure 3A and Figure 3B ) or 2W PD2 ( Figure 3C and Figure 3D ). Each symbol represents the titer from an individual animal. Bars represent the geometric mean titer (GMT). Figure 3E and Figure 3F Show the results of the ICS assay, which measures the cells expressing RSV preF A+B F-specific IFN-γ within CD4 + T cells and CD8 + T cells. Bars and error bars depict the median and interquartile range. NT: Not tested.

[0059] Figure 4 Schematically depict the wild-type (WT) RSV F protein (RSV WT) and mutant RSV F protein constructs, where "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 portion of the protein spanning the cell membrane, "CT" refers to the cytoplasmic tail peptide sequence corresponding to the portion of the protein extending into the cell cytoplasm, and "extracellular domain" refers to the peptide sequence corresponding to the portion of the protein extending into the extracellular space, where the extracellular domain contains amino acid residues 1-513 (without TM and CT, denoted by "ΔTM&CT"). The amino acid positions of each part (i.e., SP, F2, pep27, F1) or the mutations of each construct are indicated, e.g., the SP of each construct spans amino acid residues 1-25 of each construct.

[0060]

Embodiment

[0061] The present invention provides an RNA molecule (e.g., an RNA polynucleotide) that comprises 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 polypeptide comprises the 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 containing the RNA nucleic acid sequence of Table 3. In some embodiments, the RNA molecule comprises at least one of a 5' cap, 5′UTR, 3'UTR, and polyA tail. In other embodiments, the RNA molecule comprises at least one of a 5' cap, 3′UTR, and polyA tail. The present invention provides an RNA molecule that comprises a modified nucleotide (e.g., a modified RNA; modRNA).

[0062] The present invention provides an immunogenic composition that comprises any one of the RNA molecules encoding the RSV polypeptides described herein, wherein the RNA molecule is complexed with, encapsulated in, or formulated with one or more lipids to form a lipid nanoparticle (RNA-LNP). The present invention further provides an immunogenic composition that comprises any one of the RNA molecules containing at least one RNA nucleic acid described herein, wherein the RNA molecule is complexed with, encapsulated in one or more lipids, or formulated with one or more lipids to form an RNA-LNP. The present invention further provides a method for preventing, treating, or ameliorating an infection, disease, or condition (e.g., an RSV infection-related respiratory disorder, including pneumonia and bronchitis) in an individual by administering to the individual an effective amount of the RNA molecule, RNA-LNP, or immunogenic composition described herein. The present invention further provides the use of the RNA molecule, RNA-LNP, and / or immunogenic composition described herein as a vaccine.

[0063] The present invention can be more readily understood by reference to the following detailed description of embodiments of the invention and the examples included therein. It should be understood that the present invention is not limited to a particular method of manufacture, which can of course vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0064] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0065] 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 herein by reference in its entirety.

[0066]

I. Embodiments of Definitions

[0067] Unless otherwise defined 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.

[0068] Throughout this application, the terms “about” and “substantially” and “essentially” are used according to their ordinary and general meanings in the field of cell and molecular biology to indicate a deviation of ±10% of one or more values connected thereto. Thus, in any disclosed embodiment, the term may be replaced by “within [percentage] of” the specified content. In a non-limiting embodiment, the percentage includes 0.1%, 0.5%, 1%, 5% and 10%.

[0069] The recitation of a range of values herein is merely intended to be a shorthand method of individually referring to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0070] When used in conjunction with the term “comprising”, the use of the phrase “a” or “an” may mean “one”, but it is also consistent with the meanings of “one or more”, “at least one” and “one or more than one”.

[0071] The phrase “and / or” means “and” or “or”. For example, A, B and / or C includes: A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. In other words, “and / or” acts as an inclusive or operation.

[0072] The phrase “substantially all” is defined as “at least 95%”; if substantially all members of a group have a certain characteristic, then at least 95% of the members of the group have that characteristic. In some embodiments, substantially all means that members of the group having the characteristic are equal to any one of the following, at least any one of the following, or between any two of the following: 95%, 96%, 97%, 98%, 99% or 100%.

[0073] A composition and its method of use can "comprise", "consist essentially of", or "consist of": any of the components or steps disclosed throughout this specification. Throughout this specification, unless the context otherwise requires, the word "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") is inclusive or open-ended and should be understood to imply the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Having considered this, the embodiments described herein in the context of the term "comprising" can also be implemented in the context of the terms "consisting of" or "consisting essentially of". A composition and method "consisting essentially of" any of the disclosed components or steps limits the scope of the patent claim to the specified materials or steps that do not materially affect the basic and novel features of the claimed invention. The phrase "consisting of" (and any form of consisting of, such as "consist of" and "consists of") means including and being limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or essential and that no other elements can be present.

[0074] References throughout this specification to "one embodiment", "an embodiment", "a particular embodiment", "related embodiments", "an embodiment", "an additional embodiment", or "another embodiment" or combinations thereof mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the foregoing phrases 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.

[0075] The terms "inhibiting", "decreasing", or "reducing" or any variations of these terms include any measurable decrease (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease) or complete inhibition to achieve the desired result. The terms "improve", "promote", or "increase" or any variations of these terms include any measurable increase (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% increase) to achieve the desired result or yield of a protein or molecule.

[0076] As used herein, the terms "reference", "standard", or "control" describe a value against which a comparison is made. For example, a medicament, individual, population, sample, or value of interest is compared to a reference, standard, or control medicament, individual, population, sample, or value of interest. The reference, standard, or control may be tested and / or assayed and / or determined substantially contemporaneously with, and / or together with, the medicament, individual, population, sample, or value of interest being assayed, and / or may be assayed or characterized under conditions or circumstances comparable to those of the medicament, individual, population, sample, or value of interest being evaluated.

[0077] The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of cellular material, bacterial material, viral material, or medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when synthesized chemically) from its source of origin. Additionally, an isolated compound is a compound that can be administered to an individual in the isolated compound form; in other words, if a compound is attached to a column or embedded in an agarose gel, it cannot simply be considered "isolated". Further, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not naturally exist as a fragment and / or is not normally in a functional state and / or has been altered or removed from its natural state by human intervention. For example, DNA that naturally occurs in a living animal is not "isolated", but synthetic DNA or DNA that has been partially or completely separated from its coexisting materials in its natural state is "isolated". Isolated nucleic acids can exist in a substantially purified form or can exist in a non-natural environment (such as a cell into which the nucleic acid has been delivered).

[0078] As used herein, "nucleic acid" is a molecule that contains a nucleic acid moiety and refers to a DNA or RNA molecule. It may be used interchangeably with the term "polynucleotide". A nucleic acid molecule is a polymer that contains nucleotide monomers or is composed of nucleotide monomers, and the nucleotide monomers are covalently linked to each other by phosphodiester bonds 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. Nucleic acids can exist in various forms, such as: isolated segments incorporated into a sequence and recombinant vectors or recombinant polynucleotides encoding polypeptides (such as one or both chains of an antigen or antibody, or fragments, derivatives, mutant proteins, or variants thereof); polynucleotides sufficient to serve as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting the expression of the polynucleotides, mRNAs, saRNAs, modRNAs, and complementary sequences described hereinbefore. The nucleic acid may encode an epitope to which an antibody can bind.

[0079] The term "epitope" refers to the portion specifically recognized by the binding component of an immunoglobulin (such as an antibody or receptor). In some embodiments, an epitope comprises multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (such as being linearized).

[0080] The nucleic acid can be single-stranded or double-stranded and can comprise RNA and / or DNA nucleotides and their artificial variants (such as peptide nucleic acids). In some cases, a nucleic acid sequence can encode a polypeptide sequence with other heterologous coding sequences, for example, to achieve purification, transport, secretion, post-translational modification of the polypeptide, or to achieve therapeutic benefits, such as targeting or efficacy. Tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide different from the modified polypeptide.

[0081] The term "polynucleotide" refers to a nucleic acid molecule, which can be recombinant or isolated from total genomic nucleic acids. Included within the term "polynucleotide" are oligonucleotides (nucleic acids with a length of 100 residues or fewer residues), recombinant vectors (including, for example, plasmids, cosmids, phages, viruses), etc. In certain embodiments, the polynucleotide includes regulatory sequences substantially separated from its naturally occurring gene or protein-coding sequence. The polynucleotide can be single-stranded (coding or antisense) or double-stranded and can be RNA, DNA (genomic, cDNA, or synthetic), its analogs, or combinations thereof. Additional coding or non-coding sequences may (but need not) be present within the polynucleotide.

[0082] In certain embodiments, there are polynucleotide variants that have substantial identity to the sequences disclosed herein; those having sequence identity equal to any of the following, at least any of the following, at most any of the following, or between any two of the following, compared to the polynucleotide sequences provided herein, using the methods described herein (e.g., BLAST analysis using standard parameters): 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher. In certain embodiments, an isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90% identity to the amino acid sequences 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 the amino acid sequences described herein over the entire sequence length; or a nucleotide sequence complementary to the isolated polynucleotide.

[0083] Irrespective of the length of the coding sequence itself, a nucleic acid segment can be combined with other nucleic acid sequences such as promoters, polyadenylation signals, other restriction enzyme sites, polylinker sites, other coding segments, etc., such that the overall length can vary significantly. The nucleic acid can be of any length. The length of the nucleic acid can be, for example, equal to any of the following, at least any of the following, at most any 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; and / or can contain one or more additional sequences (e.g., regulatory sequences); and / or can be part of a larger nucleic acid (e.g., a vector). Thus, nucleic acid fragments of almost any length can be considered, where the total length is limited by ease of preparation and use in the intended recombinant nucleic acid scheme.

[0084] 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 skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments, the expression of which can or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. Nucleic acids encoding all or part of a polypeptide can contain contiguous nucleic acid sequences encoding all or part of such polypeptide. It is also contemplated that a particular polypeptide can be encoded by nucleic acids having varying nucleic acid sequences that have slightly different nucleic acid sequences but still encode the same or substantially similar polypeptides.

[0085] 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 can be a transcript. In some embodiments, the gene product can be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0086] Generally, the term "engineered" refers to embodiments that have been artificially manipulated. For example, a polynucleotide is considered "engineered" when two or more sequences that are not linked together in nature are artificially manipulated to be directly linked to each other in the engineered polynucleotide, and / or when a particular residue in the polynucleotide is not naturally occurring and / or is caused by the action of man to be linked to an entity or moiety to which it is not linked in nature.

[0087] 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, which are composed of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and are polymerized by a characteristic backbone structure. The backbone structure is typically formed by a phosphodiester bond between the sugar moiety (e.g., deoxyribose) of the nucleotide of a first adjacent monomer and the phosphate moiety of a second adjacent monomer. The particular sequence of monomers (e.g., the sequence of bases attached to the sugar / phosphate backbone) is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand are typically hybridized to the nucleotides of the second strand, for example, by A / T base pairing and G / C base pairing. DNA can comprise all or most of the deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide that lacks a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. Without any limitation, DNA can encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

[0088] 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 a phosphodiester bond 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, by transcription of a DNA sequence within a cell. In eukaryotic cells, transcription generally occurs in the nucleus or mitochondria. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). Processing of immature RNA in eukaryotic organisms, for example, involves various post-transcriptional modifications such as splicing, 5′ capping, polyadenylation, and export from the nucleus or mitochondria. The processed mature messenger RNA provides a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. Mature mRNA can contain a 5′ cap, 5'UTR, open reading frame, 3′UTR, and polyadenylate tail sequence. RNA can comprise all or most ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of β-D-ribofuranosyl. In one embodiment, the RNA can be messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is known to those skilled in the art, mRNA generally contains 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).

[0089] "Isolated RNA" is defined as an RNA molecule that can be recombinant or isolated from total genomic nucleic acid. An isolated RNA molecule or protein can exist in a substantially purified form or can be present in a non-natural environment such as a host cell.

[0090] "Modified RNA" or "modRNA" refers to an RNA molecule that has at least one addition, deletion, substitution, and / or alteration of one or more nucleotides compared to a naturally occurring RNA. Such alterations can refer to adding non-nucleotide material to internal RNA nucleotides, or adding to one or more 5' and / or 3' ends of the RNA. In one embodiment, such modRNA contains at least one modified nucleotide, such as an alteration of the base of a nucleotide. For example, the modified nucleotide can replace one or more uridine and / or cytidine nucleotides. For example, these replacements can be made for every instance of uridine and / or cytidine in the RNA sequence, or can be made only for selected uridine and / or cytidine nucleotides. Such alterations of the standard nucleotides in RNA can include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide in an RNA sequence can be replaced with N1-methylpseudouridine. Other such altered nucleotides are known to those skilled in the art. Such altered RNA molecules are considered analogs of naturally occurring RNA. In some embodiments, the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In some embodiments, the RNA can be a replicon RNA (replicon), particularly a self-replicating RNA or a self-amplifying RNA (saRNA).

[0091] As covered herein, without any 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 include administering the RNA described herein to a mammal, such as a human. For example, in one embodiment, such methods using RNA include an RNA vaccine encoding an antigen to induce stable neutralizing antibodies and an accompanying T cell response, thereby achieving protective vaccination. In some embodiments, a minimal vaccine dose is administered to induce robust neutralizing antibodies and an accompanying T cell response to achieve protective vaccination. In one embodiment, the RNA administered is in vitro transcribed RNA. For example, such RNA can be used to encode at least one antigen designed to generate an immune response in the mammal. A 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 that can be treated with the RNA disclosed herein include, but are not limited to, those caused by and / or affected by viral infections. Such viruses include, but are not limited to, RSV.

[0092] As used herein, "Prevent" or "prevention", when used in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more features or symptoms of the disease, disorder, or condition. Prevention is considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period.

[0093] As will be understood from the context, the "risk" of a disease, disorder, and / or condition refers to the likelihood that a particular individual will develop the disease, disorder, and / or condition. In some embodiments, the 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 the risk associated with a reference sample or 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 a particular individual. In some embodiments, the risk can reflect one or more genetic attributes, e.g., it can predispose an individual to develop (or not develop) a particular disease, disorder, and / or condition. In some embodiments, the risk can reflect one or more epigenetic events or attributes and / or one or more lifestyle or environmental events or attributes. Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition is an individual who has a higher risk of developing the disease, disorder, and / or condition than a member of the general public. In some embodiments, an individual who is 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 who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0094] The terms "protein", "polypeptide", or "peptide" are used interchangeably herein and refer to polymers of amino acid monomers, e.g., molecules comprising at least two amino acid residues. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, xenologs, orthologs, fragments, and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or can be a multimolecular complex, such as a dimer, trimer, or tetramer. A protein comprises one or more peptides or polypeptides and can fold into a three-dimensional form that may be required for the protein to perform its biological function.

[0095] As used herein, the terms "wild-type" or "WT" or "native" refer to the endogenous form of a molecule that occurs naturally in an organism. In some embodiments, the wild-type form of a protein or polypeptide is 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.

[0096] A "modified protein" or "modified polypeptide" or "variant" is a protein or polypeptide whose chemical structure, particularly 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 a protein or polypeptide can have multiple activities or functions). In particular, it is contemplated that the modified / variant protein or polypeptide can be altered with respect to one activity or function but retains wild-type activity or function, such as immunogenicity, in other embodiments. When a protein is specifically referred to herein, it generally refers to a native (wild-type) or recombinant (modified) protein. A protein can be isolated directly from a natural organism, produced by recombinant DNA / exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors that have a nucleic acid sequence encoding a polypeptide (e.g., an antigen or a fragment thereof). The term "recombinant" can be used in conjunction with the name of a polypeptide or a particular polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or as a replication product of such a molecule.

[0097] The term "fragment" of a reference amino acid sequence (peptide or protein) refers to a part of an amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and / or C-terminus. A 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. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5′-end of the open reading frame, provided that the truncated open reading frame contains a start codon for initiating translation. Fragments of an amino acid sequence contain, 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, at most, exactly the following or between any two of the following sequence identities with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived: 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%.

[0098] 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 with 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 with 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 with 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 with 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 with 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 with 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 with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived.

[0099] 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 with a reference molecule but is structurally different from that reference molecule, e.g., different in the presence or absence of one or more chemical moieties or in the amount of one or more chemical moieties compared to the reference entity. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of its structural identity with 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 characteristic structural components with the reference molecule but is different from the reference molecule in at least one aspect. 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 the amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently associated with the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits at least, at most, exactly, or between any two of the following overall sequence identities with a reference polypeptide or nucleic acid: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence component with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid exhibits a decrease in one or more levels of biological activity compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered 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 alterations at specific positions. Preferably, a variant polypeptide or nucleic acid sequence has at least one modification, e.g., 1 to about 20 modifications, compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to a reference polypeptide or nucleic acid sequence. In one embodiment, a variant polypeptide or nucleic acid sequence has 1 to about 4 modifications compared to a reference polypeptide or nucleic acid sequence.Typically, compared to a reference, fewer 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. Typically, relative to the reference, the variant polypeptide or nucleic acid contains a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of functional residues (e.g., residues involved in a particular biological activity) that are substituted, inserted, or deleted. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains fewer 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 fewer than about 5, about 4, about 3, or about 2 additions or deletions. In some embodiments, compared to the reference, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and in some embodiments, contains no addition or deletion.

[0100] In some embodiments, the reference polypeptide or nucleic acid is a "wild-type" or "WT" or "native" sequence that occurs in nature, including allelic variations. A wild-type polypeptide or nucleic acid sequence has a sequence that has not been deliberately modified. For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. A "variant" of a nucleotide sequence includes 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, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid or nucleic acid sequence.

[0101] Changes can be introduced into a nucleic acid by mutation, which can cause a change in the amino acid sequence of the polypeptide (e.g., an antigen or antibody or antibody derivative) encoded by the nucleic acid. Mutations can be introduced using any technique known in the art. In one embodiment, site-directed mutagenesis protocols, for example, are used to change one or more specific amino acid residues. In another embodiment, random mutagenesis protocols, for example, are used to change one or more randomly selected residues. In some embodiments, regardless of how it is done, mutant polypeptides can be expressed and screened for desired properties.

[0102] 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 can be made that cause amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations that selectively alter the biological activity of the polypeptide encoded by the nucleic acid can be introduced into the nucleic acid. For example, the mutations can alter the biological activity quantitatively or qualitatively. Examples of quantitative alterations include increasing, decreasing, or eliminating activity. Examples of qualitative alterations include changing the antigenic specificity of an antibody.

[0103] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.

[0104] The terms "percent identical (% identical)", "percent identity (% identity)", or like terms are intended to refer, in particular, to the percentage of nucleotides or amino acids that are identical in the best alignment between the sequences to be compared. This percentage is purely statistical, and the differences between two sequences may or may not be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is typically carried out by comparing the sequences relative to a comparison segment or "window" after the best alignment, in order to identify local regions of the corresponding sequences. The best alignment for comparison can be carried out manually or by means of the local homology algorithm of Smith & Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search algorithm of Pearson & Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by means of computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). In some embodiments, the percentage identity of two sequences is determined using the BLASTN or BLASTP algorithms available on the website of the United States National Center for Biotechnology Information (NCBI).

[0105] The percentage identity is obtained by determining the number of corresponding identical positions in the sequences being compared, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying the result by 100.

[0106] In some embodiments, the degree of similarity or identity is given for at least, at most, exactly, or between any two of the following regions of the entire length of the reference sequence: 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 for at least, at most, exactly, or between any two of the following nucleotides (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.

[0107] Homologous amino acid sequences can exhibit identity of amino acid residues of at least, at most, exactly, or between any two of the following: 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%. In one embodiment, the homologous amino acid sequence exhibits at least 95% identity of amino acid residues. In one embodiment, the homologous amino acid sequence exhibits at least 98% identity of amino acid residues. In one embodiment, the homologous amino acid sequence exhibits at least 99% identity of amino acid residues.

[0108] A fragment or variant of an amino acid sequence (peptide or protein) can be a "functional fragment" or "functional variant". The terms "functional fragment" or "functional variant" of an amino acid sequence refer to any fragment or variant that exhibits one or more functional properties that are the same as or similar to those of the amino acid sequence from which it is derived, e.g., it is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities presented by the amino acid sequence from which the fragment or variant is derived. As used herein, the terms "functional fragment" or "functional variant" particularly refer to a variant molecule or sequence that contains an amino acid sequence that has one or more amino acid changes compared to the parental molecule or sequence and is still able to perform one or more functions of the parental molecule or sequence, e.g., induce an immune response. In one embodiment, the modifications in the amino acid sequence of the parental molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. The term "mutant" of the wild-type RSV F protein, "mutant" of the RSV F protein, "RSV F protein mutant", or "modified RSV F protein" refers to a polypeptide that presents the introduced mutation relative to the wild-type F protein and is immunogenic against the wild-type F protein.

[0109] An amino acid sequence (peptide, protein or polypeptide) "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 specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to that specific sequence or a fragment thereof. An amino acid sequence derived from a specific amino acid sequence may be a variant of that specific sequence or a fragment thereof. For example, those of ordinary skill in the art will generally understand that an antigen applicable herein can 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.

[0110] In the present invention, a vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector can be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence containing an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, transfer vectors. A vector can 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. Generally, an expression vector will contain the desired coding sequence and the appropriate other sequences necessary for the coding sequence to be operably linked for expression in a particular host organism (e.g., bacteria, yeast, plants, insects or mammals) or in an in vitro expression system. A cloning vector is typically used to engineer and amplify a desired fragment (usually a DNA fragment) and may lack the functional sequences required to express one or more of the desired fragments.

[0111] As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. A pharmaceutical composition can be an immunogenic composition. In some embodiments, the active agent is present in an amount suitable for administration in a unit dose regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population. In some embodiments, the pharmaceutical composition can be formulated specifically for parenteral administration, for example, in the form of a sterile solution or suspension, or a sustained release formulation, such as by subcutaneous, intramuscular, intravenous or epidural injection.

[0112] As used herein, the term "vaccination" refers to the administration of an immunogenic composition designed to generate an immune response, such as a disease-related (e.g., pathogenic) agent (e.g., a virus). In some embodiments, vaccination can be administered before, during, and / or after exposure to a disease-related agent, and in certain embodiments, shortly before, during, and / or after exposure to the agent. In some embodiments, vaccination includes multiple administrations of a vaccine composition spaced appropriately 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, the vaccination is "personalized" in that it is directed in part or in whole against one or more epitopes (e.g., it can be or include one or more neoepitopes) determined to be present in the tumor of a particular individual.

[0113] An immune response refers to a humoral response, a cellular response, or both a humoral response and a cellular response in an organism. An immune response can be analytically measured by, including but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and / or assays that measure the regulation of the activity or expression of one or more cytokines.

[0114] As used herein, the term "combination therapy" refers to those situations where 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 can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of the first regimen are administered before any dose of the second regimen); in some embodiments, such agents are administered in an overlapping administration scheme. In some embodiments, the "administration" of combination therapy can involve administering one or more agents or one or more modalities to an individual who is receiving one or more other agents or one or more modalities in combination form. For clarity, combination therapy does not require that the individual agents be administered together (or even necessarily simultaneously) in a single composition, but in some embodiments, two or more agents or their active moieties can be administered together in a combined composition or even in the form of a combined compound (e.g., as part of a single chemical complex or covalent entity).

[0115] Those skilled in the art will appreciate that the term "administration regimen" can be used to refer to a set of unit doses (usually more than one) administered individually to an individual, which are usually spaced apart over a period of time. In some embodiments, a given therapeutic agent has a recommended administration regimen, which may involve one or more doses. In some embodiments, an administration regimen comprises a plurality of doses, each of which is temporally separated from the other doses. In some embodiments, the individual doses are separated from each other by the same length of time period; in some embodiments, an administration regimen comprises a plurality of doses and at least two different time periods separating the individual doses. In some embodiments, all the doses within an administration regimen have the same amount of unit dose. In some embodiments, the different doses within an administration regimen have different amounts. In some embodiments, an administration regimen comprises a first dose of a first amount, followed by one or more additional doses of a second amount different from the first amount. In some embodiments, an administration regimen comprises a first dose of a first amount, followed by one or more additional doses of a second amount the same as the first amount. In some embodiments, an administration regimen is associated with a desired or beneficial outcome (e.g., a therapeutic administration regimen) when administered across a relevant population.

[0116]

II. Respiratory Syncytial Virus (RSV)

[0117] The present invention provides RNA molecules (e.g., RNA polynucleotides) that comprise at least one open reading frame encoding a respiratory syncytial virus (RSV) polypeptide. The present invention further provides an immunogenic composition that comprises at least one RNA molecule encoding an RSV polypeptide, which is complexed with, encapsulated within, or formulated with one or more lipids and forms a lipid nanoparticle (LNP). The RSV polypeptide included in the immunogenic composition disclosed herein can be any RSV F protein in a pre-fusion conformation.

[0118] The term "pre-fusion conformation" refers to the structural conformation adopted by an RSV F protein or mutant thereof that can be specifically bound by the antibody D25 or AM22 when the RSV F protein or mutant is in monomeric or trimeric form, or by the antibody AM14 when the RSV F protein mutant is in trimeric form. The pre-fusion trimeric conformation is a subset of the pre-fusion conformation. As used herein, an RSV F protein or polypeptide or mutant thereof in a pre-fusion conformation can be designated as "RSV preF".

[0119] 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 AM14. The native F protein adopts the post-fusion conformation after fusion of the viral envelope with the host cell membrane. When extracted from the membrane, when expressed as an extracellular domain, or after storage, the RSV F protein can also adopt the post-fusion conformation in the context of a fusion event, such as under stress conditions (such as heating and low osmotic pressure). The term "AM14" refers to the antibody described in WO 2008 / 147196 A2, which is incorporated herein by reference in its entirety. The term "AM22" refers to the 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 / 147196 A2, which is incorporated herein by reference in its entirety.

[0120] In some embodiments, the RSV F protein is the RSV F protein of subtype A. In some embodiments, the RSV F protein is the RSV F protein of subtype B. As used herein, the terms "subtype" and "subgroup" may be used interchangeably. As used herein, the term "strain" refers to a specific isolate within each subtype or subgroup. In some embodiments, the RSV F protein is a mutant of the 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 presents an introduced mutation in the amino acid sequence relative to the amino acid sequence of the corresponding wild-type RSV F protein and is immunogenic against the wild-type RSV F protein in the pre-fusion conformation or against a virus comprising the wild-type F protein. Relative to the wild-type RSV F protein, the amino acid mutations in the mutant include amino acid substitutions, deletions, or additions.

[0121] In some embodiments, the RSV F protein is the RSV protein mutant described in WO2017 / 109629, which is incorporated herein by reference in its entirety.

[0122] In some embodiments, the RSV F protein is a mutant of the wild-type RSV F protein, wherein the introduced amino acid mutations are a pair of amino acid residues in the wild-type RSV F protein mutated to a pair of cysteines ("engineered disulfide bond mutations"). The introduced pair of cysteine residues allows the formation of a disulfide bond between the cysteine residues, which stabilizes the conformation or oligomeric state of the protein, such as the pre-fusion conformation. Examples of specific such mutant pairs 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.

[0123] In other embodiments, the RSV F protein mutant comprises amino acid mutations as one or more cavity-filling mutations. Examples of amino acids that can be target-substituted for cavity filling include small aliphatic (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr), as well as amino acids buried in the pre-fusion conformation but exposed to the solvent in the post-fusion conformation. Examples of substituting 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:

[0124] (1) S at position 55, 62, 155, 190, or 290 is substituted with I, Y, L, H, or M;

[0125] (2) T at position 54, 58, 189, 219, or 397 is substituted with I, Y, L, H, or M;

[0126] (3) G at position 151 is substituted with A or H;

[0127] (4) A at position 147 or 298 is substituted with I, L, H, or M;

[0128] (5) V at position 164, 187, 192, 207, 220, 296, 300, or 495 is substituted with I, Y, H; and

[0129] (6) R at position 106 is substituted with W.

[0130] In some specific embodiments, the RSV F protein mutant comprises at least one cavity-filling mutation selected from the group consisting of: T54H, S190I, and V296I.

[0131] In other embodiments, the RSV F protein mutant comprises an electrostatic mutation that reduces ionic repulsion or increases ionic attraction between residues in proteins that are close to each other in the folded structure. In certain embodiments, the RSV F protein mutant comprises an electrostatic substitution that reduces repulsive ionic interactions or increases attractive ionic interactions with the acidic residues of Glu487 and Asp489 from another protomer of the RSV F trimer. In some specific embodiments, the RSV F protein mutant comprises an electrostatic mutation selected from the group consisting of:

[0132] (1) E at position 82, 92 or 487 is substituted with D, F, Q, T, S, L or H;

[0133] (2) K at position 315, 394 or 399 is substituted with F, M, R, S, L, I, Q or T;

[0134] (3) D at position 392, 486 or 489 is substituted with H, S, N, T or P; and

[0135] (4) R at position 106 or 339 is substituted with F, Q, N or W.

[0136] In other embodiments, the RSV F protein mutant comprises a combination of two or more different types of mutations selected from: engineered disulfide mutations, cavity filling mutations, and electrostatic mutations. In some specific embodiments, the RSV F protein mutant comprises 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:

[0137] (1) A combination of A103C, I148C, S190I and D486S;

[0138] (2) A combination of T54H, S55C, L188C and D486S;

[0139] (3) A combination of T54H, A103C, I148C, S190I, V296I and D486S;

[0140] (4) A combination of T54H, S55C, L142C, L188C, V296I and N371C;

[0141] (5) A combination of S55C, L188C and D486S;

[0142] (6) A combination of T54H, S55C, L188C and S190I;

[0143] (7) A combination of S55C, L188C, S190I and D486S;

[0144] (8) The combination of T54H, S55C, L188C, S190I and D486S;

[0145] (9) The combination of S155C, S190I, S290C and D486S;

[0146] (10) The combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q and D489S;

[0147] (11) The combination of T54H, S155C, S190I, S290C and V296I; and

[0148] (12) The combination of S155C, S190F, S290C and V207L.

[0149] In some embodiments, the RSV F protein has subtype A and comprises the mutations S155C, S190F, S290C and V207L.

[0150] In some embodiments, the RSV F protein has subtype B and comprises the mutations S155C, S190F, S290C and V207L.

[0151] In some embodiments, the RSV F protein has subtype A and comprises the mutations S155C, S190F and S290C.

[0152] In some embodiments, the RSV F protein has subtype B and comprises the mutations S155C, S190F and S290C.

[0153] In some embodiments, the RSV F protein has subtype A and comprises the mutations A103C, I148C, S190I and D486S.

[0154] In some embodiments, the RSV F protein has subtype B and comprises the mutations A103C, I148C, S190I and D486S.

[0155] In some embodiments, the RSV F protein has subtype A and comprises the mutations T54H, A103C, I148C, S190I and D486S.

[0156] In some embodiments, the RSV F protein has subtype B and comprises the mutations T54H, A103C, I148C, S190I and D486S.

[0157] In some embodiments, the RSV F protein has subtype A and comprises the mutations T54H, S55C, L188C and D486S.

[0158] In some embodiments, the RSV F protein has subtype B and comprises the mutations T54H, S55C, L188C, and D486S.

[0159] Given the substantial conservation of the RSV F sequence, one of ordinary skill in the art can readily compare the amino acid positions between different native RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, in almost all of the identified native RSV F0 precursor proteins, the furin cleavage site falls within the same amino acid positions. Thus, the conservation of the native RSV F protein sequences across strains and subtypes allows the use of a reference RSV F sequence for comparing the 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 by reference to the amino acid sequence of the full-length native F precursor polypeptide of the RSV A2 strain; corresponding to GenInfo identifier GI 138251 and Swiss Prot identifier P03420 (SEQ ID NO: 1).

[0160] In some embodiments, the RSV F protein is in the mature form of the RSV F protein, which comprises two individual polypeptide chains, namely the F1 polypeptide and the 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 can be used. Examples of such linkers include G, GG, GGG, GS, and SAIG linker sequences. The linker can also be the full-length pep27 sequence or a fragment thereof, and the full-length pep27 sequence corresponds to the amino acids at positions 110-136 of SEQ ID NO: 1.

[0161] The F1 polypeptide chain of the mutant can have the same length as the full-length F1 polypeptide of the corresponding wild-type RSV F protein; however, it can also have deletions, such as deletions of 1 to 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 the amino acid positions 137-574 of the native RSV F0 precursor (SEQ ID NO: 1) and includes (from the N-terminus to the 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 starting from amino acid residue 514 in the native F1 polypeptide sequence is the 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.

[0162] In some embodiments, the F1 polypeptide of the RSV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks the cytoplasmic domain and part or all of the transmembrane domain. In some specific embodiments, the mutant comprises an F1 polypeptide in which the amino acid residues from position 510, 511, 512, 513, 514, 515, 520, 525 or 530 to 574 are absent. Generally, for mutants linked to a trimeric domain such as a foldon, amino acids 514 to 574 may be absent. Thus, in some specific embodiments, amino acid residues 514 to 574 are absent from the F1 polypeptide of the mutant. In other specific embodiments, the F1 polypeptide of the RSV F mutant comprises or consists of: amino acid residues 137 to 513 of the native F0 polypeptide sequence (SEQ ID NO: 1), such as the RSV 847A-foldon polypeptide (SEQ ID NO: 74); or any of the alternative F0 precursor sequences, such as those disclosed in SEQ ID NOs: 1, 2, 4, 6 and 81 to 270 of WO2017109629, which is incorporated herein by reference in its entirety.

[0163] The F1 and F2 polypeptides of the RSV F protein mutant in which one or more mutations are introduced can be from any wild-type RSV F protein known or to be discovered in the art, including but not limited to RSV subtype A and subtype B strains (including A2 Ontario and Buenos Aires) or the amino acid sequences of the F proteins of any other subtype. In some embodiments, the RSV F mutant comprises an F1 and / or F2 polypeptide from RSV A 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: 1, 2, 4, 6 and 81 to 270 of WO2017109629, which is incorporated herein by reference in its entirety. In some other embodiments, the RSV F mutant comprises an F1 and / or F2 polypeptide from RSV B 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: 2 and 211 to 263 of WO2017 / 109629, which is incorporated herein by reference in its entirety. In other embodiments, the RSV F mutant comprises an F1 and / or F2 polypeptide from 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 to 270 of WO2017109629, which is incorporated herein by reference in its entirety.

[0164] The term "F0 polypeptide" (F0) refers to the precursor polypeptide of the RSV F protein, which is composed 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 known RSV strains consists of 574 amino acids.

[0165] The term "F1 polypeptide" (F1) refers to the polypeptide chain of the mature RSV F protein. Native F1 includes approximately residues 137 - 574 of the RSV F0 precursor and consists of (from the N-terminus to the C-terminus): an extracellular region (approximately residues 137 - 524), a transmembrane domain ("TM") (approximately residues 525 - 550), and a cytoplasmic tail region ("CT") (approximately residues 551 - 574). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides that include modifications from the native sequence (e.g., amino acid substitutions, insertions, or deletions), such as modifications designed to stabilize an RSV F protein mutant or enhance the immunogenicity of an RSV F protein mutant.

[0166] The term "F2 polypeptide" (F2) refers to the polypeptide chain of the mature RSV F protein. Native F2 includes approximately residues 26 - 109 of the RSV F0 precursor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides that include modifications from the native sequence (e.g., amino acid substitutions, insertions, or deletions), such as modifications designed to stabilize an RSV F protein mutant in a pre-fusion conformation or enhance the immunogenicity of an RSV F protein mutant. 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 "foldon domain" refers to an amino acid sequence capable of forming a trimer. One example of such a foldon domain is a peptide sequence derived from bacteriophage T4 fibritin, which has the sequence GYIPEAPRDG QAYVRKDGEW VLLSTFL (SEQ ID NO: 45).

[0167] In some embodiments, the RNA molecule encodes an RSV F protein mutant disclosed in: 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. The RSV F proteins disclosed in these references are incorporated herein by reference in their entirety.

[0168] Antibodies against the RSV F protein are prevalent 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.

[0169] 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.

[0170] 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 the RSV polypeptide. In some embodiments, the RSV polypeptide is a fragment of the RSV polypeptide.

[0171] 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.

[0172] 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.

[0173] In some embodiments, the RNA molecule encodes the 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 one 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, at most, exactly, or between any two of the following identities with any one of the amino acid sequences in Table 1 (e.g., any one of SEQ ID NOs: 1-6 and 71-74): 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 one of the amino acid sequences in Table 1 (e.g., any one of SEQ ID NOs: 1-6 and 71-74).

[0174] In some embodiments, the RNA molecule sequence is transcribed from the DNA nucleic acid sequences (DNA polynucleotides) in 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 an ORF transcribed from a nucleic acid sequence that may have at least, at most, exactly, or between any two of the following identities with any one of the nucleic acid sequences in Table 2 (e.g., any one of SEQ ID NOs: 7-10 and 59-62): 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 RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any one of the nucleic acid sequences in Table 2 (e.g., any one of SEQ ID NOs: 7-10 and 59-62).

[0175] In some embodiments, the RNA molecule comprises an ORF containing the RNA nucleic acid sequence (RNA polynucleotide) of Table 3 (see Example 6). In some embodiments, the RNA molecule comprises an ORF containing a nucleic acid sequence or a fragment or variant thereof of any one of SEQ ID NOs: 11-16 and 63-70. In some embodiments, the RNA molecule comprises an ORF containing a nucleic acid sequence having at least, at most, exactly the following or any identity between any two of the following with respect to any one of the RNA nucleic acid sequences of Table 3 (e.g., any one of SEQ ID NOs: 11-16 and 63-70): 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 RNA molecule comprises an ORF containing 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).

[0176] In some embodiments, the RNA molecule comprises a stabilized RNA. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced with N1-methylpseudouridine. In some embodiments, the RNA molecule comprises a sequence in which all uridines are replaced with N1-methylpseudouridine (denoted as "Ψ"). In some embodiments, the RNA molecule comprises an ORF containing a nucleic acid sequence of any one of SEQ ID NOs: 11-16 and 63-70, wherein all uridines have been replaced with N1-methylpseudouridine (denoted as "Ψ").

[0177] In some embodiments, the RNA molecule comprises an open reading frame encoding an RSV F protein amino acid sequence that is at least, at most, exactly the following or any identity between any two of the following with respect to any one of the RSV F protein sequences of SEQ ID NOs: 1-6 and 71-74 (Table 1) or other pre-fusion RSV 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 RSV F protein amino acid sequence consisting of any one of the RSV F protein sequences of SEQ ID NOs: 1-6 and 71-74 (Table 1) or other pre-fusion RSV F proteins described herein.

[0178] In some embodiments, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence that is at least, at most, exactly, or between any two of the following identical to any one of the nucleic acid sequences of SEQ ID NOs: 7-10 and 59-62 (Table 2) or other nucleic acids 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 transcribed from a DNA nucleic acid sequence consisting of any one of the nucleic acid sequences of SEQ ID NOs: 7-10 and 59-62 (Table 2) or other nucleic acids described herein.

[0179] In some embodiments, the RNA molecule comprises an open reading frame containing an RNA nucleic acid sequence that is at least, at most, exactly, or between any two of the following identical to any one of the nucleic acid sequences of SEQ ID NOs: 11-16 and 63-70 (Table 3) or other nucleic acids 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 containing 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 containing a nucleic acid sequence of any one of SEQ ID NOs: 11-16 and 63-70 (Table 3), wherein all uridines have been replaced with N1-methylpseudouridine (denoted as "Ψ").

[0180] [[III. RNA Molecule]]

[0181] 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 comprises at least one open reading frame (ORF) encoding at least one peptide or polypeptide. The open reading frame contains a codon sequence that can be translated into a peptide or protein. The coding RNA molecule can include one (monocistronic), two (bicistronic), or more (polycistronic) ORFs, which can be codon sequences that can be translated into the polypeptide or protein of interest.

[0182] The coding RNA molecule can 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 present 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 can be a coding RNA molecule.

[0183] The RNA molecule can encode one polypeptide of interest or more, such as an antigen or more than one antigen, for example two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively or additionally, an RNA molecule can also encode more than one polypeptide of interest, such as an antigen, for example a bicistronic or tricistronic RNA molecule encoding different or the same antigens.

[0184] The sequence of the RNA molecule can be codon-optimized or de-optimized for expression in a desired host, such as a human cell. In some embodiments, compared with the wild-type coding sequence, the gene of interest (such as an antigen) described herein is encoded by a coding sequence with codon optimization and / or an increased guanosine / cytidine (G / C) content. In some embodiments, compared with the corresponding sequence region of the wild-type coding sequence, one or more sequence regions of the coding sequence are codon-optimized and / or its G / C content is increased. In some embodiments, codon optimization and / or increasing the G / C content does not change the sequence of the encoded amino acid sequence.

[0185] Those skilled in the art should understand that the term "codon optimization" refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism without changing the amino acid sequence encoded by the nucleic acid molecule. In 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 the RNA polynucleotide described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different occurrence frequencies of tRNA molecules in the cell. Therefore, the sequence of the RNA can be modified such that codons for frequently occurring tRNA molecules are inserted at positions of "rare codons".

[0186] In some embodiments, the G / C content of the coding region of the RNA (e.g., the gene sequence of interest; open reading frame (ORF)) is increased compared to the G / C content of the corresponding coding sequence of the wild-type RNA encoding the gene of interest, wherein in some embodiments, the amino acid sequence encoded by the RNA is unmodified 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. A sequence with an increased G (guanine) / C (cytosine) content is more stable compared to a sequence with an increased A (adenosine) / U (uridine) content. Given the fact that several codons encode the same amino acid (so-called genetic code degeneracy), the codons most favorable for stability (so-called alternative codon usage) can be determined. Depending on the amino acid 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 nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U or contain a lower content of A and / or U nucleotides. Thus, in some embodiments, the G / C content of the coding region of the RNA described herein is increased by at least, at most, exactly, or between any two of the following compared to the G / C content of the coding region of the wild-type RNA: 10%, 20%, 30%, 40%, 50%, 55%, or even more. In some embodiments, the coding region of the RSV RNA described herein has 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 the RSV RNA described herein has 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 the RSV RNA described herein has 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 the RSV RNA described herein has a G / C content of about 58%, about 66%, or about 62%.

[0187] In some embodiments, the RNA molecule comprises from 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 - 100,000 nucleotides).

[0188] In some embodiments, the RNA molecule has at least, at most, exactly, or between any two of the following number of nucleotides: 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.

[0189] In some embodiments, the RNA molecule comprises at least 100 nucleotides. For example, in some embodiments, the length of the RNA is between 100 and 15,000 nucleotides; 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; between 7,000 and 25,000 nucleotides. In some embodiments, the RNA molecule has at least, at most, exactly the following or between any two of the following nucleotides: 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.

[0190] The RNA molecules of the present invention can be prepared by any method known in the art, including chemical synthesis and in vitro methods such as in vitro transcription of RNA. In some embodiments, the RNA of the present invention is prepared using in vitro transcription.

[0191] In some embodiments, the RNA molecules of the present invention are purified, for example, by filtration, which can be carried out via, for example, ultrafiltration, diafiltration or, for example, tangential flow ultrafiltration / diafiltration.

[0192] In some embodiments, the RNA molecules of the present invention are lyophilized to be temperature-stable.

[0193] In some embodiments of the present invention, the RNA is or comprises messenger RNA (mRNA), which is related to an RNA transcript encoding a polypeptide. In some embodiments, the RNA disclosed herein comprises: a 5' cap, which comprises the 5' cap disclosed herein; a 5' untranslated region (5'UTR), which comprises a cap-proximal sequence; a sequence encoding a protein (such as a polypeptide) (such as the RSV pre-fusion F protein); a 3' untranslated region (3'UTR); and / or a polyadenylation (polyA) sequence.

[0194] In some embodiments, the RNA disclosed herein comprises the following components in a 5' to 3' orientation: a 5′ cap, which comprises the 5′ cap disclosed herein; a 5' untranslated region (5'UTR), which comprises a cap-proximal sequence; a sequence encoding a protein (such as a polypeptide) (such as the RSV pre-fusion F protein); a 3′ untranslated region (3′UTR); and a polyA sequence.

[0195] In some embodiments, the RNA disclosed herein further comprises a signal peptide. Non-limiting examples of signal peptides and the 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.

[0196] In some embodiments, the RNA disclosed herein encodes an antigenic fusion protein. Thus, one or more of the encoded antigens can comprise two or more proteins (such as proteins and / or protein fragments) joined together. Alternatively, the protein fused to the protein antigen does not promote a strong immune response against itself, but rather promotes a strong immune response against the antigen. In some embodiments, the antigenic fusion protein retains the functional properties of each of the original proteins. In some embodiments, the RNA disclosed herein encodes a fusion protein comprising an antigen linked to a backbone portion. 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 portions and linkers can be found, for example, in WO 2022 / 067010, the disclosure of which is incorporated herein by reference in its entirety.

[0197]

A. Modified nucleobases

[0198] In some embodiments of the present invention, the RNA molecule is unmodified chemically and comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine. In some embodiments, the nucleotides and nucleosides of the present 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 present invention comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, and / or dT).

[0199] In other embodiments of the present invention, the RNA molecule may comprise modified nucleobases, which may be incorporated into modified nucleosides and nucleotides. In some embodiments, the RNA molecule may comprise one or more modified nucleotides. In some embodiments, the RNA molecule may comprise one or more modified nucleotides. Modifications of naturally occurring nucleotides are known in the art. In some embodiments, the RNA molecule may comprise modified nucleotides.Non-limiting examples of modified nucleotides that may be included in an RNA molecule include pseudouridine, N1-methylpseudouridine, 5-methyluridine, 3-methyl-uridine, 5-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-allylamino-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid, methyl uridine 5-oxyacetate, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, methyl 5-carboxyhydroxymethyl-uridine, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine, 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine, 1-tauromethyl-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-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, α-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-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, any other modified uridine known in the art, or combinations 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.

[0200] Modifications that can be present in RNA molecules further include, but are not limited to, for example, the following: ms2io6A (2-methylthio-(N6-(cis-hydroxyisopentenyl)adenosine)); ms2m6A (2-methylthio-N6-methyladenosine); ms2t6A 2-methylthio-N6-threonylcarbamoyladenosine; g6A (N6-glycylcarbamoyladenosine); i6A (N6-isopentenyladenosine); m6A (N6-methyladenosine); t6A (N6-threonylcarbamoyladenosine); m'Am (1,2'-O-dimethyladenosine); m1A (1-methyladenosine); 2'-O-methyladenosine; Ar(p) (2'-O-ribosyladenosine (phosphate)); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; ms2hn6A (2-methylthio-N6-hydroxy-norvalylcarbamoyladenosine); 2-O-methyladenosine; Am (2-1-O-methyladenosine); 2'-O-ribosyladenosine (phosphate); isopentenyladenosine; io6A N6-(cis-hydroxyisopentenyl)adenosine; m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,2'-O-trimethyladenosine); m62A (N6,N6-dimethyladenosine); ac6A (N6-acetyladenosine); hn6A (N6-hydroxy-norvalylcarbamoyladenosine); m6t6A (N6-methyl-N6-threonylcarbamoyladenosine); m2A (2-methyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); 7-deaza-adenosine; N1-methyl-adenosine; N6,N6-(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2-(amino)adenine; 2-(aminopropyl)adenine; 2-(methylthio)-N6-(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2'-azido-2'-deoxy-ATP; 2'-deoxy-2'-a-aminoadenosine TP; 2'-deoxy-2'-a-azidoadenosine TP; 6-(alkyl)adenine; 6-(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7-(deaza)adenine; 8-(enyl)adenine; 8-(ynyl)adenine; 8-(amino)adenine; 8-(thioalkyl)adenine; 8-(enyl)adenine; 8-(alkyl)adenine; 8-(ynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxy)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; 8-oxo-adenine; azidoadenine; deazaadenine; N6-(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-azido-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'-α-Ethynyladenosine TP; 2-Aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-α-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2′-β-Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-β-Trifluoromethyladenosine TP; 2-Chloroadenosine TP; 2'-Deoxy-2',2'-difluoroadenosine TP; 2'-Deoxy-2'-α-mercaptoadenosine TP; 2'-Deoxy-2'-α-thiomethoxyadenosine TP; 2'-Deoxy-2′-β-aminoadenosine TP; 2′-Deoxy-2'-β-azidoadenosine TP; 2′-Deoxy-2'-β-bromoadenosine TP; 2'-Deoxy-2'-β-chloroadenosine TP; 2'-Deoxy-2'-β-fluoroadenosine TP; 2'-Deoxy-2'-β-iodoadenosine TP; 2'-Deoxy-2'-β-mercaptoadenosine TP; 2'-Deoxy-2′-β-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-Methoxy-adenine; 2-Methylthio-adenine; 2-Trifluoromethyladenosine TP; 3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP; 3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP; 3-Deazaadenosine TP; 4'-Azidoadenosine TP; 4′-Carbocyclic adenosine TP; 4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP; 8-Bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-Deazaadenosine TP; 2-Aminopurine; Substituted 7-Deazapurines; 7-Deaza-7-substituted purines; 7-Deaza-8-substituted purines; 7-Deaza-2,6-diaminopurine; 7-Deaza-8-aza-2,6-diaminopurine; 7-Deaza-8-aza-2-aminopurine; 2,4-Diaminopurine; 2,6-Diaminopurine; 7-Deaza-8-aza-adenine; 7-Deaza-2-aminopurine; 8-Azapurine; s2C (2-Thiocytidine); m3C (3-Methylcytidine); f5C (5-Formylcytidine); hm5C (5-Hydroxymethylcytidine); m5C (5-Methylcytidine); ac4C (N4-Acetylcytidine); Cm (2′-O-Methylcytidine); m5Cm (5,2'-O-Dimethylcytidine); f5Cm (5-Formyl-2′-O-Methylcytidine); k2C (Lysidine); m4Cm (N4,2′-O-Dimethylcytidine); ac4Cm (N4-Acetyl-2′-O-Methylcytidine); m4C (N4-Methylcytidine); N4,N4-Dimethyl-2′-OMe-cytidine TP; 4-Methylcytidine; 5-Aza-cytidine; Pseudoisocytidine; Pyrrolo-cytidine; α-Thio-cytidine;2-(thio)cytosine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-α-amino-cytidine TP; 2'-deoxy-2'-α-azido-cytidine 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-(propargyl)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-(propargyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propargylcytosine; 6-(azo)cytosine; 6-aza-cytidine; azacytosine; deazacytosine; N4-(acetyl)cytosine; 1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'-fluoro-N4-Bz-cytidine TP; 2'-fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; 2'-α-ethynylcytidine TP; 2'-α-trifluoromethylcytidine TP; 2'-β-ethynylcytidine TP; 2'-β-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-α-mercapto-cytidine TP; 2'-deoxy-2'-α-thiomethoxy-cytidine TP; 2'-deoxy-2'-β-amino-cytidine TP; 2'-deoxy-2'-β-azido-cytidine TP; 2'-deoxy-2'-β-bromo-cytidine TP; 2'-deoxy-2'-β-chloro-cytidine TP; 2'-deoxy-2'-β-fluoro-cytidine TP; 2'-deoxy-2'-β-iodo-cytidine TP; 2'-deoxy-2'-β-mercapto-cytidine TP; 2'-deoxy-2'-β-thiomethoxy-cytidine TP; 2'-O-methyl-5-(1-propargyl)cytidine TP; 3'-ethynylcytidine TP; 4'-azidocytidine TP; 4'-carbocyclic cytidine TP; 4'-ethynylcytidine TP; 5-(1-propargyl)-ara-cytidine TP; 5-(2-chloro-phenyl)-2-thiocytidine TP;5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynyl-arabinocytidine TP; 5-Ethynylcytidine TP; 5'-Hyper-cytidine TP; 5-Methoxycytidine TP; 5-Trifluoromethyl-cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; mimG (Methylguanosine); m7G (7-Methylguanosine); m2Gm (N2,2′-O-Dimethylguanosine); m2G (N2-Methylguanosine); imG (Wyosine); m1Gm (1,2'-O-Dimethylguanosine); m1G (1-Methylguanosine); 2'-O-Methylguanosine; 2'-O-Ribosylguanosine (phosphate); Gm (2'-O-Methylguanosine); Gr(p) (2'-O-Ribosylguanosine (phosphate)); preQi (7-Aminomethyl-7-deazaguanosine); preQo (7-Cyano-7-deazaguanosine); G* (Archaeosine); Methylwyosine; m2'7G (N2,7-Dimethylguanosine); m22Gm (N2,N2,2'-O-Trimethylguanosine); m2′2'7G (N2,N2,7-Trimethylguanosine); m22G (N2,N2-Dimethylguanosine); N2,7,2′-O-Trimethylguanosine; 6-Thio-guanosine; 7-Deaza-guanosine; 8-Oxo-guanosine; N1-Methyl-guanosine; a-Thio-guanosine; 2-(Propyl)guanine; 2-(Alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2′-Azido-2'-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP; 2′-Deoxy-2′-a-azidoguanosine TP; N2-Dimethylguanine; 6-(Methyl)guanine; 6-(Alkyl)guanine; 6-(Methyl)guanine; 6-Methyl-guanosine; 6-Thioguanine; 7-(Alkyl)guanine; 7-Deaza-7-substituted guanine; 7-Deaza-7-(C2-C6) alkynylguanine; 7-Deaza-8-substituted guanine; 7-(Methyl)guanine; 7-(Alkyl)guanine; 7-(Deaza)guanine; 7-(Methyl)guanine; 8-Azaguanine; 8-Hydroxyguanine; 8-Oxoguanine; 8-(Alkyl)guanine; 8-(Alkynyl)guanine; 8-(Halo)guanine; 8-(Thioalkyl)guanine; 8-(Alkenyl)guanine; 8-(Alkyl)guanine; 8-(Alkynyl)guanine; 8-(Amino)guanine; 8-(Halo)guanine; 8-(Hydroxy)guanine; 8-(Thioalkyl)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-azaguanosine; 6-Thio-7-deazaguanosine; 6-Thio-7-methylguanosine; 7-Deaza-8-azaguanosine;7-Methyl-8-oxoguanosine; N2,N2-Dimethyl-6-thioguanosine; N2-Methyl-6-thioguanosine; 1-me-GTP; 2′-Fluoro-N2-isobutyl-guanosine TP; 2'-O-Methyl-N2-isobutyl-guanosine TP; 2'-α-Ethynylguanosine TP; 2'-α-Trifluoromethylguanosine TP; 2′-β-Ethynylguanosine TP; 2'-β-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2'-difluoroguanosine TP; 2′-Deoxy-2'-α-mercapto-guanosine TP; 2′-Deoxy-2'-α-thiomethoxy-guanosine TP; 2′-Deoxy-2′-β-amino-guanosine TP; 2′-Deoxy-2′-β-azido-guanosine TP; 2'-Deoxy-2'-β-bromo-guanosine TP; 2′-Deoxy-2′-β-chloro-guanosine TP; 2'-Deoxy-2'-β-fluoro-guanosine TP; 2'-Deoxy-2′-β-iodo-guanosine TP; 2′-Deoxy-2′-β-mercapto-guanosine TP; 2′-Deoxy-2′-β-thiomethoxy-guanosine TP; 4′-Azidoguanosine TP; 4'-Carbocyclicguanosine TP; 4'-Ethynylguanosine TP; 5′-Hyper-guanosine TP; 8-Bromo-guanosine TP; 9-Deazaguanosine TP; N2-Isobutyl-guanosine TP; miI (1-Methylinosine); I (Inosine); m'lm (1,2'-O-Dimethylinosine); 2'-O-Methylinosine; 7-Methylinosine; Tm (2'-O-Methylinosine); oQ (Epoxy-Q nucleoside (queuosine)); galQ (Galactosyl-Q nucleoside); manQ (Mannosyl-Q nucleoside); Q (Q nucleoside); Allylamino-thymidine; Azidothymidine; Deazathymidine; Deoxy-thymidine; 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-Taurinomethyluridine; D (Dihydrouridine); Pseudouridine; acp3U (3-(3-Amino-3-carboxypropyl)uridine); 1-Methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-Methylpseudouridine; 1-Ethyl-pseudouridine; 2'-O-Methyluridine; 2'-O-Methylpseudouridine; 2′-O-Methyluridine; s2Um (2-Thio-2'-O-methyluridine); 3-(3-Amino-3-carboxypropyl)uridine; m3Um (3,2′-O-Dimethyluridine); 3-Methyl-pseudo-uridine TP; s4U (4-Thiouridine); chm5U (5-(Carboxyhydroxymethyl)uridine); mchm5U (5-(Carboxyhydroxymethyl)uridine methyl ester); m5Um (5,2'-O-Dimethyluridine); 5,6-Dihydro-uridine; nm5s2U (5-Aminomethyl-2-thiouridine); ncm5Um (5-Carbamoylmethyl-2'-O-methyluridine); ncm5U (5-Carbamoylmethyluridine);5-carboxyhydroxymethyluridine; methyl 5-carboxyhydroxymethyluridine; cnmm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); 5-carboxymethylaminomethyluridine; cmnm5U (5-carboxymethylaminomethyluridine); 5-carbamoylmethyluridine TP; mcm5Um (5-methoxycarbonylmethyl-2′-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); mcm5U (5-methoxycarbonylmethyluridine); mo5U (5-methoxyuridine); m5s2U (5-methyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); m5D (5-methyldihydrouridine); 5-oxoacetic acid-uridine TP; 5-oxoacetic acid-methyl ester-uridine TP; dihydrouracil; pseudouracil; N1-methyl-pseudouracil; N1-ethyl-pseudouracil; cmo5U (uridine 5-oxoacetate); mcmo5U (methyl ester of uridine 5-oxoacetate); 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(isopentenylaminomethyl)-2-thiouridine TP; 5-(isopentenylaminomethyl)-2'-O-methyluridine TP; 5-(isopentenylaminomethyl)uridine TP; 5-propynyluracil; a-thio-uridine; 1-(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-pseudouracil; 1-(aminocarbonylvinyl)-2(thio)-pseudouracil; 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil; 1-(aminocarbonylvinyl)-4-(thio)pseudouracil; 1-(aminocarbonylvinyl)-pseudouracil; 1-substituted 2(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4-(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylaminocarbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-methyl-pseudo-UTP; 1-ethyl-pseudo-UTP; 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'-α-amino uridine TP; 2′-Deoxy-2'-α-azido uridine TP; 2-Methylpseudouridine; 3-(3-Amino-3-carboxypropyl)uracil; 4-(Thio)pseudouracil; 4-(Thio)pseudouracil; 4-(Thio)uracil; 4-Thiouracil; 5-Aminouracil; 5-(1,3-Diazol-1-ylalkyl)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-(thio)uracil; 5-(Methylaminomethyl)-2,4-(dithio)uracil; 5-(Methylaminomethyl)-4-(thio)uracil; 5-(Propargyl)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)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-ylalkyl)uracil; 5-(Methoxy)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-(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-(Propargyl)uracil; 5-(Trifluoromethyl)uracil; 5-Aminoallyl-uridine; 5-Bromo-uridine; 5-Iodo-uridine; 5-Uracil; 6-(Azido)uracil; 6-(Azido)uracil; 6-Aza-uridine; Allylamino-uracil; Azauracil; Deazauracil; 5-Methyluracil; 5-(Hydroxymethyl)uracil; 5-Chlorouracil; 5-Fluorouracil; 5-Bromouracil; N3-(Methyl)uracil; Pseudouridine-1-2-acetic acid; Pseudouracil; 4-Thio-pseudouridine triphosphate; 1-Carboxymethyl-pseudouridine; 1-Methyl-1-deaza-pseudouridine;1-Propynyl-uridine; 1-Taurinomethyl-1-methyl-uridine; 1-Taurinomethyl-4-thio-uridine; 1-Taurinomethyl-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-dihydropseudouridine; 2-Thio-dihydrouridine; 2-Thio-pseudouridine; 4-Methoxy-2-thio-pseudouridine; 4-Methoxy-pseudouridine; 4-Thio-1-methyl-pseudouridine; 4-Thio-pseudouridine; 5-Aza-uridine; Dihydropseudouridine; (±)1-(2-Hydroxypropyl)pseudouridine TP; (2R)-1-(2-Hydroxypropyl)pseudouridine TP; (2S)-1-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2-Bromo-vinyl)arabinouridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)arabinouridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; 1-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; 1-(2,2-Diethoxyethyl)pseudouridine TP; 1-(2,4,6-Trimethylbenzyl)pseudouridine TP; 1-(2,4,6-Trimethyl-benzyl)pseudo-UTP; 1-(2,4,6-Trimethyl-phenyl)pseudo-UTP; 1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP; 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)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP; 1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Aminobenzyl)pseudo-UTP; 1-(4-Aminobutyl)pseudo-UTP; 11(4-Aminophenyl)pseudo-UTP; 1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP; 1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP; 1-(4-Iodobenzyl)pseudouridine TP; 1-(4-Methanesulfonylbenzyl)pseudouridine TP; 1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP; 1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP; 1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP; 1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP; 1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP; 1-(4-Trifluoromethylbenzyl)pseudouridine TP; 1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propanoyl]pseudouridine TP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propanoyl}pseudouridine TP; 1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP; 1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP; 1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP; 1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP; 1-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-Homoallylpseudouridine TP; 1-Hydroxymethylpseudouridine TP; 1-Isopropyl-pseudo-UTP; 1-me-2-thio-pseudo-UTP; 1-me-4-thio-pseudo-UTP; 1-me-α-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; 1-Methyl-6-(2,2,2-trifluoroethyl)pseudo-UTP; 1-Methyl-6-(4-(N-morpholino))-pseudo-UTP; 1-Methyl-6-(4-thio(N-morpholino))-pseudo-UTP; 1-Methyl-6-(substituted phenyl)pseudo-UTP; 1-Methyl-6-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-carboxyethyl-pseudo-UTP; 1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP; 1-Methyl-6-formyl-pseudo-UTP;1-Methyl-6-hydroxyamino-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-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP; 1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP; 1-Methyl-6-trifluoromethoxy-pseudo-UTP; 1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-(N-Morpholinyl)methylpseudouridine TP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-Trimethylacetylpseudouridine TP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-Propynyl-pseudouridine; 1-p-Tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP; 1-Thiomethoxymethylpseudouridine TP; 1-Thio(N-morpholinyl)methylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'-Anhydro-uridine TP; 2'-Bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-me-UTP; 2'-OMe-pseudo-UTP; 2'-α-Ethynyluridine TP; 2'-α-Trifluoromethyluridine TP; 2′-β-Ethynyluridine TP; 2'-β-Trifluoromethyluridine TP; 2′-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-α-mercaptouridine TP; 2'-Deoxy-2′-α-thiomethoxyuridine TP; 2'-Deoxy-2'-β-aminouridine TP; 2'-Deoxy-2'-β-azidouridine TP; 2'-Deoxy-2'-β-bromouridine TP; 2'-Deoxy-2'-β-chlorouridine TP; 2'-Deoxy-2'-β-fluorouridine TP; 2'-Deoxy-2'-β-iodouridine TP; 2'-Deoxy-2'-β-mercaptouridine TP; 2'-Deoxy-2'-β-thiomethoxyuridine TP; 2-Methoxy-4-thio-uridine; 2-Methoxyuridine; 2'-O-Methyl-5-(1-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclicuridine TP; 4'-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furyl)uridine TP; 5-Cyano-uridine TP; 5-Dimethylamino-uridine TP; 5'-homo-Uridine TP; 5-Iodo-2'-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteriomethyl-6-deutero-uridine TP; 5-Trifluoromethyl-uridine TP; 5-Vinylarabinouridine TP; 6-(2,2,2-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-Ethylcarboxylate-pseudo-UTP; 6-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-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP; 6-Tert-butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6-Trifluoromethyl-pseudo-UTP; α-Thio-pseudo-UTP; Pseudouridine 1-(4-Methylbenzenesulfonate)TP; Pseudouridine 1-(4-Methylbenzoate)TP; Pseudouridine TP 1-[3-(2-Ethoxy)]propionate; Pseudouridine TP 1-[3-{2-(2-[2-(2-Ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionate; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-Ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionate; Pseudouridine TP 1-[3-{2-(2-[2-Ethoxy]-ethoxy)-ethoxy}]propionate; Pseudouridine TP 1-[3-{2-(2-Ethoxy)-ethoxy}]propionate; Pseudouridine TP 1-Methylphosphonate; Pseudouridine TP 1-Diethylmethylphosphonate; Pseudo-UTP-N1-3-Propionate; Pseudo-UTP-N1-4-Butyrate; Pseudo-UTP-N1-5-Valerate; Pseudo-UTP-N1-6-Hexanoate; Pseudo-UTP-N1-7-Heptanoate; Pseudo-UTP-N1-Methyl-p-benzoate; Pseudo-UTP-N1-p-Benzoate; yW (Wybutosine); OHyW (Hydroxywybutosine); imG2 (Isowybutosine); o2yW (Peroxywybutosine); OHyW* (Under-modified hydroxywybutosine); imG-14 (4-Demethylwybutosine); 2,6-(Diamino)purine; 1-(Aza)-2-(Thio)-3-(Aza)-phenoxazin-1-yl; 1,3-(Diaza)-2-(Oxo)-phenothiazin-1-yl; 1,3-(Diaza)-2-(Oxo)-phenoxazin-1-yl; 1,3,5-(Triaza)-2,6-(Dioxo)-naphthalene; 2-(Amino)purine; 2,4,5-(Trimethyl)phenyl; 2'-Methyl, 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-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)carbostyrilyl; 3-(methyl)carbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)carbostyrilyl; 5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloropurine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)phenoxazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)phenothiazin-1-yl; 7-(aminoalkylhydroxy)-1,3-(diaz)-2-(oxo)phenoxazin-1-yl; 7-(aza)indolyl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz)-2-(oxo)phenoxazin-1-yl; 7-(guanidylalkyl-hydroxy)-1,3-(diaz)-2-(oxo)phenothiazin-1-yl; 7-(guanidylalkylhydroxy)-1,3-(diaz)-2-(oxo)phenoxazin-1-yl; 7-(propynyl)carbostyrilyl; 7-(propynyl)carbostyrilyl; propynyl-7-(aza)indolyl; 7-deazainosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)phenoxazin-1-yl; 7-substituted 1,3-(diaz)-2-(oxo)phenoxazin-1-yl; 9-(methyl)imidazopyridyl; aminoindolyl; anthryl; bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-o-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridyl; inosinyl; carbostyrilyl; isoguanisine; N2-substituted purine; N6-methyl-2-aminopurine; N6-substituted purine; N-alkylated derivative; naphthyl;Nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purines; O-alkylated derivatives; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; para-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; condensed pentaphenyl; propylene-anthracenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidinyl; pyrrolopyrazinyl; stilbenzyl; substituted 1,2,4-triazole; tetracenyl; tubercidine; xanthine; xanthosine-5'-TP; 2-thio-zeacrine; 5-aza-2-thio-zeacrine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-aminoriboside-TP; formycin A TP; formycin B TP; pyrrolosine TP; 2'-OH-arabinonucleoside adenosine TP; 2′-OH-arabinonucleoside cytidine TP; 2'-OH-arabinonucleoside uridine TP; 2′-OH-arabinonucleoside guanosine TP; 5-(2-methoxycarbonylvinyl)uridine TP; N6-(19-amino-pentaoxanonadecyl)adenosine TP; hydrogen (abasic residue); 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 aforementioned modified nucleobases. In some embodiments, 1, 2, 3, 4, 5, or more of the aforementioned modifications may be excluded from the RNA molecules disclosed herein.;

[0201] In some embodiments, the modified nucleobases in the RNA molecule include pseudouridine (ψ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4'-thiouridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2,6-diaminopurine, inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-lysidine, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-carbamoyloxymethyluridine, 5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-Q riboside, methylated undermodified hydroxywybutosine, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Q base, preQ0 base, preQ1 base, and combinations of two or more thereof.In some embodiments, the RNA molecule comprises a combination of at least two (e.g., two, three, four, or more) of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some embodiments, one, two, three, four, five, or more of the aforementioned modified nucleobases are excluded from the RNA molecules disclosed herein.

[0202] Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2′-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabinocytidine, 2'-F-cytidine, and 2'-OH-arabinocytidine. In some embodiments, one, two, three, four, five, or more of the aforementioned modified cytosines may be excluded from the RNA molecules disclosed herein.

[0203] In some embodiments, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 5-cyano-uridine, 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), methyl uridine 5-oxyacetate (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), methyl 5-carboxyhydroxymethyl-uridine (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnmVU), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauromethyl-uridine (xm5U), 1-tauromethyl-pseudouridine, 5-tauromethyl-2-thio-uridine (xmVu), 1-tauromethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, e.g., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydrourididine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m5Um), 2′-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm5Um), 3,2′-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)]uridine. In some embodiments, one, two, three, four, five, or more of the foregoing modified uridines may be excluded from the RNA molecules disclosed herein.,

[0204] In some embodiments of the present invention, the modified nucleotides include either N1-methylpseudouridine and / or pseudouridine.

[0205] In some embodiments, the RNA molecule comprises a nucleotide modified with N1-methylpseudouridine. In some embodiments, the RNA molecule comprises a nucleotide modified with pseudouridine.

[0206] In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises modified nucleosides in place of each uridine. In some embodiments, the RNA molecule comprises a sequence in which at least one uridine is replaced with N1-methylpseudouridine. In some embodiments, the RNA molecule comprises a sequence in which all uridines are replaced with N1-methylpseudouridine. N1-methylpseudouridine is represented as “Ψ” in the sequence. As used herein, the term “uracil” describes a nucleobase that can occur in the nucleic acid of RNA. As used herein, the term “uridine” describes a nucleoside that can occur in RNA. “Pseudouridine” is an example of a modified nucleoside, which is an isomer of uridine, in which uracil is linked to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

[0207] In some embodiments, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced by N1-methylpseudouridine and / or pseudouridine. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which at least, at most, exactly, or any two of the following (inclusive or exclusive) uridines are replaced by N1-methylpseudouridine and / or pseudouridine: 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%. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which all uridines are replaced by N1-methylpseudouridine and / or pseudouridine.

[0208] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a 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-adenosine, 7-deaza-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 (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxy-norvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxy-norvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2′-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2′-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabinofuranosyladenosine, 2'-F-adenosine, 2′-OH-arabinofuranosyladenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine. In some embodiments, one, two, three, four, five or more of the foregoing modified adenines may be excluded from the RNA molecules disclosed herein.

[0209] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxylwybutosine (o2yW), hydroxywybutosine (OhyW), under-modified hydroxywybutosine (OhyW*), 7-deazaguanosine, Q nucleoside (Q), epoxy-Q nucleoside (oQ), galactosyl-Q nucleoside (galQ), mannosyl-Q nucleoside (manQ), 7-cyano-7-deazaguanosine (preQo), 7-aminomethyl-7-deazaguanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2′7G), N2,N2,7-dimethyl-guanosine (m2'2'7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine (m2'7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arabinoguanosine, and 2'-F-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.

[0210] 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 fully (e.g., uniformly) modified along the entire length of the molecule. For example, in the polynucleotides of the invention or in a given predetermined sequence region thereof, one or more or all or a given type of nucleotide (e.g., purines and / or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified. In some embodiments, all nucleotides X in the polynucleotides of the invention (or in a given sequence region thereof) are modified nucleotides, where X can be any one of the nucleotides A, G, U, C, and / or any one 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, the polynucleotide may be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue (such as those set forth above). The modified nucleotide may be replaced with one compound having a single unique structure, or may be replaced with plural compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0211] RNA molecules can contain modified nucleotides that are or are about 1% to 100% (related to the total nucleotide content or to one or more types of nucleotides (e.g., any one or more of A, G, U, and / or C)) (e.g., at least, at most, exactly 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% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It should be understood that any remaining percentage is made up by the presence of unmodified A, G, U, and / or C.

[0212] In some embodiments, the RNA molecule can include phosphoramidate, phosphorothioate, and / or methylphosphonate linkages.

[0213] In some embodiments, an RNA molecule can include one or more structural and / or chemical modifications and / or alterations that confer desirable properties to the polynucleotide, including, in some embodiments, reduced degradation in a cell or organism and / or substantially no 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 the RNA molecule without significant chemical modification of the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, a structural modification has a chemical nature and is thus a chemical modification. However, a structural modification will result in a different nucleotide sequence. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification of the polynucleotide.

[0214] In some embodiments, a modified RNA molecule introduced into a 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 can 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.

[0215] In some embodiments, an RNA molecule can include one or more modified nucleotides in addition to any 5' cap structure. In some embodiments, an RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, except for an optionally present 5′ cap such as 7-methylguanosine, which is further described below. In some embodiments, the RNA can include a 5′ cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides can be methylated at the 2' position of the ribose.

[0216]

B. 5' Cap

[0217] 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.

[0218] In some embodiments, the 5' cap moiety is a native 5' cap. A "native 5' cap" is defined as a cap that includes 7-methylguanosine bonded to the 5' end of an mRNA molecule via a 5'-5' triphosphate bond. In some embodiments, the guanosine nucleoside included in the 5' cap may be modified, for example, by methylation at one or more positions on the base (guanine) (e.g., at the 7-position) and / or by methylation at one or more positions on the ribose. In some embodiments, the guanosine nucleoside included in the 5' cap contains 3'O methylation at the ribose (3'OMeG). In some embodiments, the guanosine nucleoside included in the 5' cap contains methylation at the 7-position of guanine (m7G). In some embodiments, the guanosine nucleoside included in the 5' cap contains methylation at the 7-position of guanine and 3'O methylation at the ribose (m7(3′OMeG)). The 5' cap may be incorporated during RNA synthesis (e.g., co-transcriptional capping), or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some embodiments, compared to co-transcriptional capping with an appropriate reference comparator, the capping efficiency of RNA is increased when co-transcriptionally capped with the caps disclosed herein. In some embodiments, increasing the capping efficiency may increase the translation efficiency and / or translation rate of the RNA, and / or increase the expression of the encoded polypeptide. In some embodiments, capping is performed after purification of the RNA molecule (e.g., tangential flow filtration).

[0219] In some embodiments, the RNA described herein comprises a 5' cap or a 5' cap analog, such as cap 0, cap 1, or cap 2. In some embodiments, the provided RNA does not have an uncapped 5'-triphosphate. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide. In some embodiments, the 5' cap moiety is a 5' cap analog. In some embodiments, the RNA can be capped with a 5' cap analog. Cap structures include but are not limited to 7 mG(5')ppp(5')N 1 pN 2 p(cap 0), 7 mG(5')ppp(5')N 1 m pNp(cap 1), and 7 mG(5')ppp(5′)N 1 m pN 2 m p(cap 2). In some embodiments, one, two, three, four, five, or more of the foregoing cap structures may be excluded from the RNA molecules disclosed herein.

[0220] In some embodiments, the RNA described herein comprises cap 0. In some embodiments, cap 0 is N7-methylguanosine, and the cap 0 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G). In some embodiments, the cap 0 structure is bonded to the RNA via a 5′-5′ triphosphate bond and is also referred to herein as m7G, m7Gppp, and / or m7G(5′)ppp(5′). The 5′ cap can be used with the structure 7 mG(5′)ppp(5′)N 1 pN 2 p(cap 0) or its derivative is methylated, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5′ cap, typically the 5′ end of mRNA. Exemplary enzymatic reactions for capping can include using vaccinia virus capping enzyme (VCE) comprising mRNA triphosphatase, guanylyltransferase, and guanine-7-methyltransferase, which catalyzes the construction of an N7-monomethylated cap 0 structure. The cap 0 structure plays an important role in maintaining the stability and translation efficacy of RNA molecules. In cells, the cap 0 structure is crucial for the efficient translation of capped mRNA.

[0221] In some embodiments, the RNA described herein comprises, for example, cap 1 as described herein. The 5′ cap of the RNA molecule can be further modified at the 2′-O position by a 2′-O-methyltransferase, which results in the production of a cap 1 structure (m7Gppp[m2′-Ο]N), which can further increase translation efficiency. In some embodiments, the cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and the first nucleotide methylated at 2′O in the RNA (2′OmeN 1 ). In some embodiments, the cap 1 structure is bonded to the RNA via a 5′-5′ triphosphate bond and is also referred to herein as m7GpppN m , where N m represents any nucleotide having 2′O methylation, 7 mG(5')ppp(5′)N 1 m pNp, m7Gppp(2′OMeN 1 ) and / or m7G(5')ppp(5′)(2'OMeN 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, m7G(5')ppp(5′)(2'OmeN 1) The cap 1 structure contains a second nucleotide N 2 , which is the cap-proximal nucleotide at position 2 and is selected from A, G, C, or U (m7G(5′)ppp(5')(2′OmeN 1 )N 2 ). 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.

[0222] In some embodiments, the cap 1 structure contains a guanosine nucleoside methylated at the 7-position of guanine (m7G), one or more additional modifications (such as methylation on the ribose), and a first nucleotide that is 2'O-methylated in the RNA. In some embodiments, the cap 1 structure contains a guanosine nucleoside methylated at the 7-position of guanine, 3'O-methylation at the ribose (m7(3'OMeG)), and a first nucleotide that is 2′O-methylated in the RNA (2'OMeN 1 ). In some embodiments, the cap 1 structure is bonded to the RNA via a 5′-5' triphosphate bond and is also referred to herein as m7(3′OMeG)ppp(2′OMeN 1 ) and / or m7(3'OMeG)(5′)ppp(5′)(2'OMeN 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'OMeG)(5')ppp(5′)(2′OMeN 1 ) cap 1 structure contains a second nucleotide N 2 , which is the cap-proximal nucleotide at position 2 and is selected from A, G, C, or U (m7(3′OMeG)(5')ppp(5′)(2′OmeN 1 )N 2 ). 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, one, two, three, four, five, or more of the foregoing cap 1 structures may be excluded from the RNA molecules disclosed herein.

[0223] 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 a cap 2. In some embodiments, a cap 1 structure comprising a second nucleotide with 2′-O-methylation is a cap 2 structure.

[0224] In some embodiments, the RNA molecule can be enzymatically capped at the 5′ end using vaccinia guanylyl transferase, guanosine triphosphate, and S-adenosyl-L-methionine to obtain a cap 0 structure. The inverted 7-methylguanosine cap is added via a 5′-5′ triphosphate bridge. Alternatively, a cap 1 structure can be obtained using a 2′-O-methyltransferase and vaccinia guanylyl transferase, wherein, in addition to the cap 0 structure, the 2′OH group on the penultimate nucleotide is methylated. S-adenosyl-L-methionine (SAM) is the cofactor used as the methyltransferase reagent. Non-limiting examples of 5′ cap structures are those that exhibit enhanced binding of cap-binding polypeptides, increased half-life, reduced sensitivity to 5′ endonucleases, and / or reduced 5′ decapping, compared to synthetic 5′ cap structures (or wild-type, native, or physiological 5′ cap structures) known in the art.

[0225] For example, recombinant vaccinia virus capping enzyme and recombinant 2′-O-methyltransferase can generate a typical 5′-5′ triphosphate linkage between the 5′ terminal nucleotide of the mRNA and the guanine cap nucleotide, where the cap guanine includes N7 methylation and the 5′ terminal nucleotide of the mRNA includes 2′-O-methyl. Such a structure is referred to as a cap 1 structure. This cap results in higher translational ability and cellular stability and reduced activation of cellular pro-inflammatory cytokines compared to other 5′ cap analog structures known in the art, for example.

[0226] Cap species can include one or more modified nucleosides and / or linker moieties. For example, a cap can include a guanine nucleotide joined by a triphosphate bond at its 5′ position and a guanine (G) nucleotide methylated at the 7 position, such as m7G(5′)ppp(5′)G, typically written as m7GpppG. The cap species can also be an anti-reverse cap analog. 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 can be excluded from the RNA molecules disclosed herein.

[0227] In some embodiments, the 5' cap includes a cap analogue, e.g., the 5' cap may include a guanine analogue. Exemplary guanine analogues 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 analogues may be excluded from the cap structures disclosed herein.

[0228] In some embodiments, the capping region may include a single cap or a series of nucleotides that form a cap. In this embodiment, the length of the capping region may be from 1 to 10, such as 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 this embodiment, the length of the capping region is at least, at most, exactly the following or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, there is no cap. In some embodiments, the lengths of the first and second operable regions may range from 3 to 40 nucleotides, such as 5 to 30, 10 to 20, 15, or at least 4 or 30 or fewer, and may contain one or more signals and / or restriction enzyme cleavage sequences in addition to the start and / or stop codons. In some embodiments, the lengths of the first and second operable regions are at least, at most, exactly the following or between any two of the following (inclusive 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 contain one or more signals and / or restriction enzyme cleavage sequences in addition to the start and / or stop codons.

[0229] Other embodiments of the 5' cap structure include, but are not limited to, glyceryl, inverted deoxyabasic residue (moiety), 4',5'-methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified base nucleotide, threo-furanosyl pentose nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety, 3'-3'-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3'-thiophosphate, dithiophosphate, and / or bridged or unbridged methylphosphonate moiety. In some embodiments, one, two, three, four, five or more of the foregoing 5' cap structures may be excluded from the RNA molecules disclosed herein.

[0230] In some embodiments, the RNA molecules of the present invention comprise at least one 5' cap structure. In some embodiments, the RNA molecules of the present invention do not comprise a 5' cap structure.

[0231] 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 Cell Metabolism Modulation in Methods in Molecular Biology 69 (Rabinovich, P. H. ed.), 2013). In one embodiment, the 5' capping structure comprises a modified 5' cap 1 structure (m 7 G + m3'-5'-ppp-5'-Am). In one embodiment, the 5' capping structure comprises (3'OMe)-m 2 7 ,3' -O Gppp(m 1 2′-O)ApG (TriLink BioTechnologies). This molecule is identical to the natural RNA cap structure as it starts with a guanosine methylated at N7 and is bonded to the first encoded nucleotide (in this case, adenosine) of the transcribed RNA by a 5'-5' triphosphate bond. This guanosine is also methylated at the 3'-hydroxyl of the ribose to mitigate possible reverse incorporation of the cap molecule. The 2'-hydroxyl of the ribose on the adenosine is methylated, conferring a cap 1 structure.

[0232]

C. Untranslated Region (UTR)

[0233] The 5' UTR is a regulatory region located at the 5' end of the protein open reading frame that is transcribed into mRNA but not translated into an amino acid sequence, and / or the corresponding region in an RNA polynucleotide (such as an mRNA molecule). Untranslated regions (UTRs) can be present at the 5' end (upstream) (5' UTR) and / or the 3' end (downstream) (3' UTR) of the open reading frame.

[0234] In some embodiments, the UTR is derived from an mRNA that is naturally abundant in a particular tissue (e.g., lymphoid tissue) targeted for mRNA expression. In some embodiments, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR can increase protein synthesis by increasing the retention time of the mRNA in the translating polyribosome (message stability) and / or the rate of ribosome initiation of translation based on the message (message translational efficiency). Thus, the UTR sequence can extend protein synthesis in a tissue-specific manner.

[0235] In some embodiments, the regulatory features of the UTR can be incorporated into the RNA of the present invention to particularly enhance the stability of the molecule. Specific features can also be incorporated to ensure controlled downregulation of the transcript in the event that it is misdirected to an undesired organ site. A variety of 5' UTR and 3' UTR sequences are known and available in the art.

[0236] It should be understood that any UTR from any gene can be incorporated into the regions of the RNA of the present invention. In addition, various wild-type UTRs of any known gene can be utilized. Artificial UTRs that are variants and not wild-type regions are also within the scope of the present invention. The placement orientation of these UTRs or portions thereof can be the same as in the transcript from which they are selected, or their orientation and / or position can vary. Thus, the 5' and / or 3' UTRs can be inverted, shortened, extended, and / or so can one or more other 5' UTRs or 3' UTRs. As used herein, the term "altered" when related to a UTR sequence means that the UTR has been changed in some way relative to a reference sequence. For example, a 5' UTR and / or 3' UTR can be altered relative to a wild-type or native UTR by changing its orientation and / or position as taught above, and / or can be altered by including additional nucleotides, deleting nucleotides, exchanging and / or transposing nucleotides. Any one of these changes results in an "altered" UTR (whether 5' and / or 3'), including variant UTRs.

[0237] In some embodiments, double, triple, or quadruple UTRs can be used, such as 5' and / or 3' UTRs. As used herein, a "double" UTR is a UTR encoded by two copies of the same UTR in tandem or substantially in tandem. For example, a double β-globin 3′ UTR can be used. Having patterned UTRs is also within the scope of the present invention. As used herein, a "patterned UTR" is those UTRs that reflect a repetitive or alternating pattern, such as AB AB AB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter A, B, or C represents a different UTR at the nucleotide level.

[0238] The RNA can encode a polypeptide of interest belonging to a protein family that is expressed in a particular cell, tissue, and / or at some time during development. In some embodiments, the UTRs from any one of these genes can be exchanged with any other UTRs of the same or different protein families to generate new RNA molecules. As used herein, "protein family" is used in the broadest sense and refers to a group of two or more polypeptides of interest that share at least one function, structure, feature, localization, origin, and / or expression pattern.

[0239] In some embodiments, the 5′UTR and 3'UTR sequences are computationally derived. In some embodiments, the 5′UTR and 3'UTR are derived from mRNAs that are naturally abundant in a tissue. The tissue can be, for example, liver, stem cells, and / or lymphoid tissue. Lymphoid tissue can include, for example, any of lymphocytes (such as B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and / or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils, and reticulocytes. In some embodiments, the 5'UTR and 3'UTR are derived from an alphavirus. In some embodiments, the 5'UTR and 3′UTR are from a wild-type alphavirus.

[0240] In some embodiments, the untranslated region can also include a translation enhancer element (TEE). As a non-limiting example, TEEs can 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.

[0241]

i. 5'UTR

[0242] In some embodiments, the RNA disclosed herein includes a 5'UTR. The 5'UTR, if present, is located at the 5' end and begins at the transcription start site upstream of the start codon of the protein-coding region. The 5'UTR is downstream of the 5′ cap, if present, for example, immediately adjacent to the 5′ cap. The 5'UTR can contain various regulatory elements, such as a 5' cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which can play a role in the control of translation initiation. The 5′UTR can contain a signature, such as a Kozak sequence, which is also involved in the process by which ribosomes initiate translation of many genes. The 5'UTR can also form a secondary structure involved in the binding of elongation factors.

[0243] In some embodiments, the 5′UTR disclosed herein includes, for example, a cap-proximal sequence disclosed herein. In some embodiments, the cap-proximal sequence includes a sequence adjacent to the 5' cap. In some embodiments, the cap-proximal sequence includes the nucleotides at positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide.

[0244] In some embodiments, the cap structure includes one or more polynucleotides of the cap-proximal sequence. In some embodiments, the cap structure includes an m7 guanosine cap and the nucleotide +1 (N 1 ) of the RNA polynucleotide. In some embodiments, the cap structure includes an m7 guanosine cap and the nucleotide +2 (N 2 ) of the RNA polynucleotide. In some embodiments, the cap structure includes an m7 guanosine cap and the nucleotides +1 and +2 (N 1 and N2 )。

[0245] Those skilled in the art reading the present invention will understand 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) can be included in the RNA by being included in a cap entity (e.g., a cap 1 structure, etc.); alternatively, in some embodiments, at least some residues of the cap proximal sequence can be added enzymatically (e.g., by a polymerase, such as T7 polymerase). For example, in those exemplary embodiments where (m 2 7,3'-O )Gppp(m 2'-O )ApG caps are utilized, the +1 and +2 residues are the (m 2 7,3'-O )A and G residues of the cap, and the +3, +4, and +5 residues are added by a polymerase (e.g., T7 polymerase).

[0246] In some embodiments, the cap proximal sequence comprises the N 1 and / or N 2 of the cap structure, where 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 the N 1 and N 2 of the cap structure as well as N 3 , N 4 and N 5 , where N 1 to N 5 correspond to positions +1, +2, +3, +4, and / or +5 of the RNA polynucleotide. In some embodiments, N 1 , N 2 , N 3 , N 4 or N 5 is any nucleotide, such as A, C, G, or U. In some embodiments, N 1 N 2Comprises any one of the following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG or UU. In some embodiments, N 1 N 2 Comprises AG, and N 3 N 4 N 5 Comprises any one of the following: 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.

[0247] In some embodiments, the cap-proximal sequence comprises N of the cap structure 1 and N 2 , and a sequence comprising A 3 A 4 X 5 (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.

[0248] In some embodiments, the cap-proximal sequence comprises N of the cap structure 1 and N 2 , and a sequence comprising C 3 A 4 X 5 (SEQ ID NO: 47; wherein X 5 is A, G, C or U), wherein N 1 and N 2Each 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.

[0249] In some embodiments, the cap proximal sequence comprises N 1 and N 2 of the cap structure, and a sequence comprising X 3 Y 4 X 5 (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 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 is A. In some embodiments, X 3 and / or X 5 is C. In some embodiments, X 3 and / or X 5 is G. In some embodiments, X 3 and / or X 5 is 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.

[0250] In some embodiments, the cap proximal sequence comprises N 1 and N 2 of the cap structure and a sequence comprising A 3 C 4 A 5The sequence of (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.

[0251] In some embodiments, the cap-proximal sequence comprises the N 1 and N 2 of the cap structure and comprises the sequence of A 3 U 4 G 5 (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.

[0252] In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned cap-proximal sequences may be excluded from the 5' UTR of the RNA molecules disclosed herein.

[0253] In some embodiments of the present invention, the 5' UTR is a heterologous UTR, for example, a UTR found in nature that binds to a different ORF. In another embodiment, the 5' UTR is a synthetic UTR, for example, a UTR that does not exist in nature. 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 the ORF, for example, binds to the ORF, such that it can function, for example, to 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 compared to a reference RNA molecule comprising a reference 5' UTR or an RNA molecule lacking a 5' UTR. In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned 5' UTR functions may be excluded.

[0254] Exemplary 5'UTRs include 5′UTRs derived from Xenopus or human alpha globin or beta globin, human cytochrome b-245a, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, CMV immediate early 1 (IE1) gene, TEV, HSP70 5', 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 a TOP gene lacking a 5'TOP motif (oligopyrimidine tract); a 5′UTR derived from the ribosomal protein large 32 (L32) gene; a 5′UTR of a 5'UTR derived from the hydroxysteroid (17p) dehydrogenase 4 gene (HSD17B4); or a 5′UTR of a 5'UTR derived from ATP5A1. In some embodiments, the 5'UTR is derived from SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421, and SEQ ID NO: 1422 of patent application WO2013 / 143700, the disclosure of which is incorporated herein by reference in its entirety, or a sequence having at least, at most, exactly, or between any two of the following (inclusive or exclusive) identity to any of the foregoing sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. The 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.

[0255] In some embodiments, the 5'UTR comprises a 5'UTR region from a gene encoding any of the following, or a sequence from its homolog, fragment, or variant: RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, 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, RPL11, 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, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB; or a gene sequence having at least, at most, exactly, or between any two of the following (inclusive or exclusive) identity with any of the foregoing gene sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. 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.

[0256] In one embodiment, the DNA encoding the 5′UTR disclosed herein comprises a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to 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 RNA disclosed herein comprises a 5′UTR comprising a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the 5′UTR provided in either SEQ ID NO: 18 or 19 (where the transcribed 5' cap structure is underlined). In one embodiment, the 5'UTR comprises the sequence of either SEQ ID NO: 18 or 19 (where the transcribed 5' cap structure is underlined).

[0257]

SEQ ID NO:17(DNA)

[0258] AG AATAAACT AGTATTCTTC TGGTCCCCAC AGACTCAGAG AGAACCC

[0259]

SEQ ID NO:18(RNA)

[0260] AG AAUAAACU AGUAUUCUUC UGGUCCCCAC AGACUCAGAG AGAACCC

[0261]

SEQ ID NO:19(RNA)

[0262] AG AAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCAC AGACΨCAGAG AGAACCC

[0263] In one embodiment, the DNA encoding the 5'UTR disclosed herein comprises a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to 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 RNA disclosed herein comprises a 5'UTR containing a sequence having at least, at most, exactly the following, or between any two of the following identity to the 5'UTR provided in either SEQ ID NO: 52 or 53: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one embodiment, the 5'UTR comprises the sequence of either SEQ ID NO: 52 or 53 (wherein the transcribed 5' cap structure is underlined).

[0264]

SEQ ID NO: 51 (DNA)

[0265] G ATAGGCGGC GCATGAGAGA AGCCCAGACC AATTACCTAC CCAAA

[0266]

SEQ ID NO: 52 (RNA)

[0267] G AUAGGCGGC GCAUGAGAGA AGCCCAGACC AAUUACCUAC CCAAA

[0268]

SEQ ID NO: 53 (RNA)

[0269] G AΨAGGCGGC GCAΨGAGAGA AGCCCAGACC AAΨΨACCΨAC CCAAA

[0270] In some embodiments, one, two, three, or more of the foregoing 5'UTR sequences may be excluded from the RNA molecules disclosed herein.

[0271]

ii. 3'UTR

[0272] In some embodiments, the RNA disclosed herein comprises a 3′UTR. The 3'UTR, if present, is located downstream of the open reading frame of the protein-coding sequence, such as downstream of the stop codon of the protein-coding region. The 3′UTR is generally the portion of the mRNA that lies between the protein-coding sequence of the mRNA and the polyA tail. Thus, in some embodiments, the 3'UTR is upstream of the polyA sequence, if present, such as immediately adjacent to the polyA sequence. The 3'UTR can be involved in regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

[0273] The native or wild-type 3'UTR contains an extension of adenosine and uridine. These AU-rich signatures are particularly prevalent in genes with higher turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three classes: Class I AREs contain several dispersed copies of the AUUUA motif within a U-rich region. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Class III AREs do not contain the AUUUA motif. Most proteins known to bind to AREs destabilize the molecule. Thus, the introduction, removal, and / or modification of 3'UTR AREs can be used to regulate the stability of the nucleic acids (such as RNA) of the present invention. When engineering a particular nucleic acid, in some embodiments, one or more copies of an ARE can be introduced to make the RNA less stable and thus reduce translation and decrease the production of the resulting protein. Similarly, in some embodiments, AREs can be identified and removed and / or mutated to increase intracellular stability and thus increase translation and the production of the resulting protein. Transfection experiments can be performed using the nucleic acids of the present invention in relevant cell lines, and protein production can be analyzed at different time points after transfection. For example, molecules can be engineered with different AREs and cells can be transfected and the proteins produced can be analyzed at 6 hours, 12 hours, 24 hours, 48 hours, and 7 days after transfection using an ELISA kit specific for the relevant protein. In some embodiments, one or more AU-rich sequences can be removed from the 3'UTR. Alternatively, the AU-rich sequences can be maintained in the 3'UTR.

[0274] The 3'UTR can also contain elements that are not encoded in the template of the transcribed RNA but are added during maturation after transcription, such as the polyA tail. The 3'UTR of the mRNA is not translated into an amino acid sequence. In some embodiments, the RNA disclosed herein comprises a 3'UTR containing an F element and / or an I element. In some embodiments, the 3'UTR or its proximal sequence contains a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an Xhol site.

[0275] In some embodiments of the present invention, the 3'UTR is a heterologous UTR, for example, a UTR found in nature that binds 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 the ORF, for example, binds to the ORF so that it can function, for example, compared to a reference RNA molecule containing a reference 3'UTR or an RNA molecule lacking a 3'UTR, to increase, enhance, stabilize, and / or extend protein production from the RNA molecule, and / or increase protein expression and / or total protein production from the RNA molecule. In some embodiments, one, two, three, four, five, or more of the foregoing 3'UTR functions may be excluded.

[0276] Exemplary 3'UTRs include: 3'UTRs derived from the following: albumin gene, α-globin gene, β-globin gene, ribosomal protein gene, tyrosine hydroxylase gene, lipoxygenase gene, and collagen α gene (such as collagen α1(1) gene); or homologs, fragments, or variants of 3'UTRs derived from genes containing the following: albumin gene, α-globin gene, β-globin gene, ribosomal protein gene, tyrosine hydroxylase gene, lipoxygenase gene, and / or collagen α gene (such as collagen α1(1) gene), SEQ ID NOs: 1369-1390 according to Patent Application WO2013 / 143700 (the disclosure of which is incorporated herein by reference in its entirety), or a sequence having at least, at most, exactly the following, or any two of the following (inclusive or exclusive) identity to any of the foregoing sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some embodiments, the sequence UUUGAAUU is used. In some embodiments, one, two, three, four, five, or more of the foregoing 3'UTR sequences may be excluded from the RNA molecules disclosed herein.

[0277] In some embodiments, the 3'UTR comprises: the sequences of transcripts, which include 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_001199802.1, 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_001199340.1, NM_001199341.1, NMJD01199342.1, NM_001199343.1, NM_001199344.1, NM_001199345.1, NM_000986.3, NM_000987.3, NM_000988.3, NM_000989.3, NM_000990.4, NM_001136134.1, NMJD00991.4, NM_001136135.1, NM_001136136.1, NM_001136137.1, 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_001011.3, 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_001146227.1, NM_001023.3, NM_001024.3, NM_001025.4, NM_001028.2, NM_001029.3, NM_001030.4, NM_002954, NM_001135592.2, NM_001177413.1, 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_001199972.1, NM_021104.1, NM_022551.2, NM_033022.3, NM_001142284.1, NM_001026.4, NM_001142285.1, NM_001142283.1, NM_001142282.1, 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_011172.1, NG_011253.1, NG_0009524, NR_002309.1, NG_010827.2, NG_009952.2, or NG_009517.1; or a sequence of a transcript having at least, at most, exactly, or between any two of the following (inclusive or exclusive) identity with any one of the foregoing transcripts: 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.

[0278] In some embodiments, the 3′UTR comprises: a sequence from the 3′UTR region of a gene encoding a ribosomal protein, such as ribosomal protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosomal protein L11 (RPL11), ribosomal protein L12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A), ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21), ribosomal protein L22 (RPL22), ribosomal protein L23a (RPL23A), ribosomal protein L17 (RPL17), ribosomal protein L24 (RPL24), ribosomal protein L26 (RPL26), ribosomal protein L27 (RPL27), ribosomal protein L30 (RPL30), ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28), ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31), ribosomal protein L32 (RPL32), ribosomal protein L35a (RPL35A), ribosomal protein L37 (RPL37), ribosomal protein L37a (RPL37A), ribosomal protein L38 (RPL38), ribosomal protein L39 (RPL39), ribosomal protein large P0 (RPLP0), ribosomal protein large P1 (RPLP1), ribosomal protein large P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A (RPS3A), X-linked ribosomal protein S4 (RPS4X), Y-linked ribosomal protein S41 (RPS4Y1), ribosomal protein S5 (RPS5), ribosomal protein S6 (RPS6), ribosomal protein S7 (RPS7), ribosomal protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S10 (RPS10), ribosomal protein S11 (RPS11), ribosomal protein S12 (RPS12), ribosomal protein S13 (RPS13), ribosomal protein S15 (RPS15), ribosomal protein S15a (RPS15A), ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19), ribosomal protein S20 (RPS20), ribosomal protein S21 (RPS21), ribosomal protein S23 (RPS23), ribosomal protein S25 (RPS25), ribosomal protein S26 (RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomal protein L15 (RPL15), ribosomal protein S2 (RPS2), ribosomal protein L14 (RPL14), ribosomal protein S14 (RPS14), ribosomal protein L10 (RPL10), ribosomal protein L10a (RPL10A), ribosomal protein L35 (RPL35), ribosomal protein L13a (RPL13A), ribosomal protein L36 (RPL36), ribosomal protein L36a (RPL36A), ribosomal protein L41 (RPL41), ribosomal protein S18 (RPS18), ribosomal protein S24 (RPS24), ribosomal protein L8 (RPL8), ribosomal protein L34 (RPL34), ribosomal protein S17 (RPS17), ribosomal protein SA (RPSA) or ribosomal protein S17 (RPS17); or a sequence of a gene encoding a ribosomal protein having at least, at most, exactly the following or any two of the following (inclusive or exclusive) identities to any one of the ribosomal gene protein sequences described above: 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.

[0279] In some embodiments, the 3'UTR comprises: a 3'UTR region from a gene encoding a ribosomal protein or a sequence from a gene comprising: ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52), Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) of far upstream element (FAU), ribosomal protein L22-like 1 (RPL22L1), ribosomal protein L39-like (RPL39L), ribosomal protein L10-like (RPL10L), ribosomal protein L36a-like (RPL36AL), ribosomal protein L3-like (RPL3L), ribosomal protein S27-like (RPS27L), ribosomal protein L26-like 1 (RPL26L1), ribosomal protein L7-like 1 (RPL7L1), ribosomal protein L13a pseudogene (RPL13AP), ribosomal protein L37a pseudogene 8 (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal protein S26 pseudogene 11 (RPS26P11), ribosomal protein L39 pseudogene 5 (RPL39P5), ribosomal protein large PO pseudogene 6 (RPLP0P6), and ribosomal protein L36 pseudogene 14 (RPL36P14); and / or a sequence of a gene encoding a protein having at least, at most, exactly, or between any two of the following identities (inclusive or exclusive) with any one of the foregoing 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.

[0280] One of ordinary skill in the art will understand that heterologous and / or synthetic 5′UTRs can be used with any desired 3′UTR sequence, and vice versa. For example, a heterologous 5′UTR can be used with a synthetic and / or heterologous 3'UTR.

[0281] In one embodiment, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to 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 RNA disclosed herein comprises a 3'UTR containing a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the 3'UTR provided in either SEQ ID NO: 21 or 22. In one embodiment, the 3'UTR comprises the sequence of either SEQ ID NO: 21 or 22.

[0282]

SEQ ID NO:20(DNA)

[0283]

[0284]

SEQ ID NO:21(RNA)

[0285]

[0286]

SEQ ID NO:22(RNA)

[0287]

[0288] In one embodiment, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to 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 RNA disclosed herein comprises a 3'UTR containing a sequence having at least, at most, exactly the following, or between any two of the following identity to the 3'UTR provided in either SEQ ID NO: 24 or 25: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one embodiment, the 3'UTR comprises the sequence of either SEQ ID NO: 24 or 25.

[0289]

SEQ ID NO:23(DNA)

[0290]

[0291]

SEQ ID NO: 24 (RNA)

[0292]

[0293]

SEQ ID NO: 25 (RNA)

[0294]

[0295] In some embodiments, one, two, three, four, five or more of the foregoing 3′UTR sequences may be excluded from the RNA molecules disclosed herein.

[0296]

D. Open Reading Frame (ORF)

[0297] The 5' and 3' UTRs are operably linked to an open reading frame (ORF), which can be a codon sequence capable of being translated into a polypeptide of interest. The open reading frame can be a sequence of several DNA or RNA nucleotide triplets that can be translated into a peptide or protein. The ORF can start with a start codon at its 5' end and the next region, such as a combination of three consecutive nucleotides that typically encodes the amino acid methionine (ATG or AUG), and its length is usually a multiple of 3 nucleotides. The open reading frame can end with at least one stop codon, which includes but is not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some embodiments, the open reading frame can end with one, two, three, four or more stop codons, said 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 can be isolated, or it can be incorporated into a longer nucleic acid sequence, such as into a vector or mRNA. The open reading frame can also be referred to as the "(protein) coding region" or "coding sequence".

[0298] As stated herein, an RNA molecule can include one (monocistronic), two (bicistronic) or more (polycistronic) open reading frames.

[0299] In some embodiments, the ORF encodes a non-structural viral gene. In some embodiments, the ORF further includes one or more subgenomic promoters. In some embodiments, the RNA molecule includes 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, while 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.

[0300] 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 the RSV F protein. In a preferred embodiment, the RNA molecule comprises at least one open reading frame encoding a respiratory syncytial virus (RSV) prefusion F protein (preF) polypeptide.

[0301]

E. Gene of interest

[0302] The RNA molecules described herein may include a gene of interest. The gene of interest encodes a polypeptide of interest. Non-limiting examples of polypeptides of interest include, for example, biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell-penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, endomembrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, polypeptides encoded by the human genome for which no therapeutic indication has been established but which still have utility in the research and exploration fields, or combinations thereof. In some embodiments, one, two, three, four, five or more of the foregoing polypeptides of interest may be excluded. The sequences of specific genes of interest can be readily identified by those skilled in the art using public and private databases (such as ).

[0303] In some embodiments, the RNA molecule includes the coding region of a gene of interest. In some embodiments, the gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or 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-epitopic. 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, one, two, three, four, five or more of the foregoing antigenic polypeptides may be excluded.

[0304] The term "antigen" may refer to a substance that can be recognized by the immune system (e.g., by the adaptive immune system) and that can, for example, induce an antigen-specific immune response, such as by the formation of antibodies and / or antigen-specific T cells, as part of the adaptive immune response. An antigen may be or may comprise a peptide or protein that can be presented by MHC to T cells. An antigen may be the translation product of a provided nucleic acid molecule (e.g., an RNA molecule comprising at least one coding sequence as described herein). Additionally, fragments, variants, and derivatives of an antigen (such as a peptide or protein) that comprise at least one epitope are understood to be antigens.

[0305] In some embodiments, RNA encoding a gene of interest (e.g., an antigen) is expressed in the cells of the individual being treated to provide the gene of interest (e.g., an antigen). In some embodiments, the RNA is transiently expressed in the cells of the individual. In some embodiments, the expression of the gene of interest (e.g., an antigen) is at the cell surface. In some embodiments, the gene of interest (e.g., an antigen) is expressed and presented in the context of MHC. In some embodiments, the gene of interest (e.g., an antigen) is expressed into the extracellular space, e.g., the antigen is secreted.

[0306] In some embodiments, the RNA molecule comprises a coding region of a gene of interest (e.g., an antigen). In some embodiments, the RNA molecule comprises a coding region of a gene of interest (e.g., an antigen) derived from a pathogen associated with an infectious disease. In some embodiments, the RNA molecule comprises a coding region of a gene of interest (e.g., an antigen) derived from RSV.

[0307] In some embodiments, the RNA molecule encodes an RSV preF protein or a fragment or variant thereof.

[0308] In some embodiments, the RNA polynucleotide or a composition or pharmaceutical formulation comprising the same described herein comprises the nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence having at least 80% identity with the nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity with the polypeptide sequence disclosed herein. In some embodiments, the RNA polynucleotide or a composition or pharmaceutical formulation comprising the same described herein is transcribed from a DNA template. In some embodiments, the DNA template for transcribing the RNA polynucleotide described herein comprises a sequence complementary to the RNA polynucleotide. In some embodiments, the gene of interest described herein is encoded by the RNA polynucleotide described herein comprising the nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide encodes a polypeptide having at least 80% identity with the polypeptide sequence disclosed herein. In some embodiments, the polypeptide described herein is encoded by an RNA polynucleotide which is transcribed from a DNA template comprising a sequence complementary to the RNA polynucleotide.

[0309] In some embodiments, the RNA molecule encodes an RSV preF protein comprising a sequence of any one of SEQ ID NOs: 1 to 6 and 71 to 74, or a fragment or variant thereof.

[0310] In some embodiments, the RNA molecule encodes an RSV preF protein synthesized from a nucleic acid sequence or a fragment or variant thereof, the nucleic acid sequence comprising any one of SEQ ID NOs: 7-10 and 59-62.

[0311]

F. Poly-A tail

[0312] In some embodiments, the RNA molecules disclosed herein comprise a polyadenylic acid (poly-A) sequence as described herein, for example. In some embodiments, the poly-A sequence is located downstream of the 3′ UTR, for example adjacent to the 3′ UTR. A “poly-A tail” or “poly-A sequence” refers to a stretch of consecutive adenine residues that can be attached to the 3′ end of an RNA molecule, which for example has up to or up to about 400 adenosine nucleotides, for example is or is about 20 - about 400, preferably is or is about 50 - about 400, more preferably is or is about 50 - about 300, even more preferably is or is about 50 - about 250, and most preferably is or is about 60 - about 250 adenosine nucleotides. Poly-A sequences are known to those skilled in the art and can be attached to the 3′ UTR in the RNA molecules described herein. The poly-A tail can increase the stability, half-life, and / or translation efficiency of the RNA molecule.

[0313] After cleavage, most pre-mRNAs acquire a polyadenylated tail, with the exception of pre-mRNAs of replication-dependent histone transcripts that end with a histone stem-loop replacing the poly-A sequence. In this context, 3′-end processing is a nuclear co-transcriptional process that facilitates the transport of mRNA from the nucleus to the cytoplasm and affects the stability and translation of mRNA. The formation of this 3′ end occurs in a two-step reaction guided by the cleavage / polyadenylation machinery and depends on the presence of two sequence elements in the mRNA precursor (pre-mRNA): the hexanucleotide polyadenylation signal and the downstream GU-rich sequence. In the first step, the pre-mRNA between these two elements is cleaved to a free 3′ hydroxyl. In the second step, the newly formed 3′ end is elongated by polyadenylation or addition of the poly-A sequence.

[0314] Polyadenylation refers to the addition of a poly-A sequence to an RNA molecule, such as to an immature mRNA. Polyadenylation can be induced by a so-called polyadenylation signal. This signal can be located near the 3'-end of the RNA molecule to be polyadenylated or within a nucleotide extension at its 3'-end. The 3'-UTR of an artificial nucleic acid molecule can also contain a polyadenylation signal. The polyadenylation signal typically contains a hexamer composed of adenine and uracil / thymine nucleotides, preferably the hexamer sequence AAUAAA, but other sequences (preferably hexamer sequences) are also conceivable. Polyadenylation usually occurs during the processing of pre-mRNA (also called immature mRNA). Generally, RNA maturation (from pre-mRNA to mature mRNA) includes a step of polyadenylation. Poly-A tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including but not limited to cloning a poly-T segment into a DNA template, or post-transcriptional addition by using a poly-A polymerase. The term can refer to the polyadenylation of RNA as a cellular process, or to polyadenylation by in vitro enzymatic reactions with suitable enzymes (such as E. coli poly-A polymerase) or by chemical synthesis.

[0315] The RNA molecules disclosed herein can have a poly-A sequence that is ligated to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription, or a poly-A sequence that is encoded by DNA and transcribed by a template-dependent RNA polymerase. In some embodiments, the poly-A sequence is ligated during RNA transcription, such as during the preparation of in vitro transcribed RNA, based on a DNA template that contains repetitive dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.

[0316] The DNA sequence (coding strand) encoding the polyA sequence is referred to as a polyA cassette. In some embodiments, the polyA cassette present in the coding strand of the DNA consists essentially of dA nucleotides, but is interspersed with a random sequence of the four nucleotides (dA, dC, dG, and dT). The length of such a random sequence can be at least, at most, exactly, 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. Such cassettes are disclosed, for example, in WO 2016 / 005324 A1, which is incorporated herein by reference. Any polyA cassette disclosed in WO 2016 / 005324 A1 can be used in the present invention. A polyA cassette consisting essentially of dA nucleotides but interspersed with a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and a length of, for example, 5 to 50 nucleotides shows plasmid DNA propagation in Escherichia coli at the DNA level and is still associated with beneficial properties related to supporting RNA stability and translation efficiency at the RNA level. In some embodiments, the polyA sequence contained in the RNA polynucleotides described herein consists essentially of adenosine nucleotides, but is interspersed with a random sequence of the four nucleotides (A, C, G, U). The length of such a random sequence can be at least, at most, exactly, 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.

[0317] The poly-A sequence can be located at any position within the 3'UTR. In some embodiments, there are no nucleotides other than adenosine nucleotides flanking the poly-A sequence on the 3' side, for example, the 3' end of the poly-A sequence is not masked by nucleotides other than adenosine or there are no nucleotides other than adenosine following the 3' end of the poly-A sequence. In some embodiments, the poly-A sequence can be located at the 3' end of the 3'UTR, for example, the 3'UTR does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide on the 3' side of the poly-A sequence; more preferably, the 3'UTR does not contain other components on the 3' side of the poly-A sequence. In some embodiments, the poly-A sequence is located at the 3' end of the RNA molecule, for example, the artificial nucleic acid molecule does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide on the 3' side of the poly-A sequence. Alternatively, the poly-A sequence can be located at the 5' end of the 3'UTR, for example, adjacent to the 3' of the ORF of the artificial nucleic acid molecule, or within the 3'UTR, for example, flanking the 5' side and 3' side of other 3'UTR components. In some embodiments, the poly-A sequence flanks the 3' side of the poly-C sequence and / or histone stem-loop sequence. Alternatively or additionally, the poly-A sequence can flank the 5' side of a 3'UTR component derived from, for example, the human albumin or globulin gene.

[0318] 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 (such as a polyadenylation signal) (such as 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 a nucleotide sequence containing at least, at most, exactly the following, or any two of the following (inclusive or exclusive): 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 nucleotides, where the nucleotide sequence preferably does not contain more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides. In some embodiments, the nucleotide sequence separating the poly-A sequence from the polyadenylation signal contains or contains approximately 1 to approximately 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 contain more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides.

[0319] 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)(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 specificity factor (CPSF) that cooperate with CstF, PAP, PAB2, CFI, and / or CFII. In some embodiments, the polyadenylation signal (e.g., the consensus sequence NNUANA) is located less than or less than about 50 nucleotides downstream of the 3' end of the 3'UTR component as defined herein, for example, at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 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 ligate a polyA sequence to the immature RNA. Thus, the resulting RNA can comprise a 3'UTR containing at least one polyA sequence, and wherein additional polyA sequences follow the 3'UTR.

[0320] The poly(A) sequence can 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 includes a poly(A) tail that comprises, consists essentially of, or consists of: a sequence that is or is about 25 to about 400 adenosine nucleotides, a sequence that is or is about 50 to about 400 adenosine nucleotides, a sequence that is or is about 50 to about 300 adenosine nucleotides, a sequence that is or is about 50 to about 250 adenosine nucleotides, a sequence that is or is about 60 to about 250 adenosine nucleotides, or a sequence that is or is about 40 to about 100 adenosine nucleotides.In some embodiments, the poly(A) tail comprises at least, at most, exactly, or any two of the following (inclusive or exclusive) adenosine nucleotides, 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.In this context, "consisting essentially of" means that most 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 nucleotides in the poly-A sequence are adenosine nucleotides, but the remaining nucleotides are allowed 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 nucleotides in the poly-A sequence) are adenosine nucleotides.

[0321] In some embodiments, the RNA molecule comprises a poly-A tail having a sequence containing more than 30 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly-A tail containing or containing approximately 40 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly-A tail containing or containing approximately 80 adenosine nucleotides. In some embodiments, the 3' poly-A tail has an extension of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some particular embodiments, the RNA molecule comprises or comprises approximately 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises or comprises approximately 80 consecutive adenosine residues. The poly-A tail can play a key regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperadenylation can signal RNA degradation.

[0322] In some embodiments, the poly-A tail can be located within the RNA molecule or other nucleic acid molecules, such as within a vector, for example within a vector that serves as a template for generating RNA (such as mRNA) by a transcription vector. In some embodiments, the RNA molecule may not include a poly-A tail.

[0323] In some embodiments, the poly-A tail can be located within the RNA molecule or other nucleic acid molecules, such as within a vector, for example within a vector that serves as a template for generating RNA (such as mRNA) by a transcription vector. In some embodiments, the RNA molecule may not include a poly-A tail.

[0324] In one embodiment, the DNA encoding the poly(A) tail disclosed herein comprises a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 26. In one embodiment, the DNA encoding the poly(A) tail comprises the sequence of SEQ ID NO: 26. In one embodiment, the RNA disclosed herein comprises a poly(A) tail containing a sequence having at least, at most, exactly the following, or between any two of the following (inclusive or exclusive): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 26. In one embodiment, the poly(A) tail comprises the sequence of SEQ ID NO: 26. In one embodiment, the poly(A) tail comprises a sequence of SEQ ID NO: 26 ± 2 adenosine (A) nucleotides. In one embodiment, the poly(A) tail comprises a sequence of SEQ ID NO: 26 ± 1 adenosine (A) nucleotides. In one embodiment, the poly(A) tail comprises the sequence of SEQ ID NO: 26. In one embodiment, the poly(A) tail comprises a sequence of SEQ ID NO: 26 ± 2 adenosine (A) nucleotides. In one embodiment, the poly(A) tail comprises a sequence of SEQ ID NO: 26 ± 1 adenosine (A) nucleotides. In some embodiments, the poly(A) tail comprises the sequence of SEQ ID NO: 26.

[0325] 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.

[0326]

SEQ ID NO: 26 (DNA, RNA)

[0327] AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA

[0328]

G. Other Components

[0329] In some embodiments of the present invention, the RNA molecule further comprises a chain-terminating nucleoside. For example, chain-terminating nucleosides can include those nucleosides that are deoxygenated at the 2' and / or 3' position of their sugar moiety. Such species can include 3'-deoxyadenosine (cordycepin), 3'-deoxyuridine, 3'-deoxycytidine, 3'-deoxyguanosine, 3'-deoxythymidine, and 2',3'-dideoxynucleosides (such as 2′,3′-dideoxyadenosine, 2′,3'-dideoxyuridine, 2',3'-dideoxycytidine, 2′,3′-dideoxyguanosine, and 2′,3′-dideoxythymidine). 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, incorporation of a chain-terminating nucleotide into an mRNA (e.g., at the 3' end) can cause stabilization of the mRNA, as described, for example, in International Patent Publication No. WO 2013 / 103659.

[0330] In some embodiments of the present invention, the RNA molecule further comprises a stem-loop, such as a histone stem-loop. The stem-loop can comprise 2, 3, 4, 5, 6, 7, 8 or more nucleobase pairs. For example, the stem-loop can comprise 4, 5, 6, 7 or 8 nucleobase pairs. The stem-loop can be located in any region of the mRNA. For example, the stem-loop can be located in the untranslated region (5'UTR or 3'UTR), coding region, or in, before, or after the polyA sequence or tail. In some embodiments, the stem-loop can affect one or more functions of the mRNA, such as initiation of translation, translation efficiency, and / or transcriptional termination. Such histone stem-loop sequences can 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.

[0331] In some embodiments, the combination of a polyA sequence or polyadenylation signal and at least one histone stem-loop (even though the two represent alternative mechanisms in nature) act synergistically to increase protein expression beyond that observed in the case of either of the individual components. In some embodiments, the synergistic effect of the combination of polyA and at least one histone stem-loop is independent of the order of the components and / or the length of the polyA sequence.

[0332] In some embodiments, the RNA does not contain a histone downstream element (HDE). HDE includes a purine-rich polynucleotide extension of approximately 15 to 20 nucleotides of the naturally occurring stem-loop 3', which represents the binding site of U7 snRNA and is involved in processing histone pre-mRNA into mature histone mRNA.

[0333] In some embodiments, the histone stem-loop generally originates from a histone gene and includes intramolecular base pairing of two adjacent or fully reverse complementary sequences separated by a spacer and consisting of short sequences that form a loop of the structure. The unpaired loop region generally cannot base pair with either of the stem-loop components. The stability of the stem-loop structure generally depends on the length of the paired region, the number of mismatches or bulges, and / or the base composition. In some embodiments, wobble base pairing (non-Watson-Crick base pairing) can occur. In some embodiments, at least one histone stem-loop sequence has a length of 15 to 45 nucleotides.

[0334] In some embodiments, the RNA molecule includes a poly(C) sequence (e.g., within the 3'UTR). In some embodiments, the polyC sequence has at least, at most, exactly, or any two of the following (inclusive or exclusive) cytidines: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some embodiments, the polyC sequence has or has approximately 30 cytidines.

[0335] In some embodiments, the RNA molecule includes an internal ribosome entry site (IRES) sequence or an IRES motif. In some embodiments, for example, if the RNA encodes two or more peptides or proteins, the IRES sequence separates the ORFs. If the RNA molecule is a dicistronic or polycistronic nucleic acid molecule, the IRES sequence may thus be applicable.

[0336] In some embodiments, the RNA does not contain introns. In some embodiments, the RNA alternatively or additionally includes microRNA binding sites.

[0337] Representative RNA molecules comprising combinations of the components disclosed herein may include, but are not limited to, the following in the 5'-to-3' direction:

[0338] ORF - polyA sequence;

[0339] ORF - IRES - ORF - polyA sequence;

[0340] ORF - 3'UTR - polyA sequence;

[0341] ORF - polyA sequence - 3'UTR;

[0342] ORF - 3'UTR - polyA sequence - poly(C) sequence - histone stem - loop;

[0343] ORF - 3′UTR - polyA sequence - poly(C) sequence - polyA sequence;

[0344] ORF - 3'UTR - polyA sequence - histone stem - loop - polyA sequence;

[0345] 5'UTR - ORF - 3'UTR;

[0346] 5'UTR - ORF - polyA sequence;

[0347] 5'UTR - ORF - polyA sequence - poly(C) sequence - histone stem - loop;

[0348] 5′UTR - ORF - polyA sequence - poly(C) sequence - polyA sequence;

[0349] 5′UTR - ORF - polyA sequence - histone stem - loop - polyA sequence;

[0350] 5'UTR - ORF - 3'UTR - polyA sequence;

[0351] 5′UTR - ORF - 3'UTR - polyA sequence - poly(C) sequence

[0352] 5'UTR - ORF - 3′UTR - polyA sequence - poly(C) sequence - histone stem - loop;

[0353] 5'-cap - 5'UTR - ORF - 3'UTR;

[0354] 5'-cap - 5'UTR - ORF - polyA sequence;

[0355] 5′-cap - 5′UTR - ORF - 3'UTR - polyA sequence;

[0356] 5'-cap - 5'UTR - ORF - 3'UTR - polyA sequence - poly(C) sequence; or

[0357] 5'-cap - 5'UTR - ORF - 3'UTR - polyA sequence - poly(C) sequence - histone stem - loop.

[0358] In some embodiments, one, two, three, four, five or more of the foregoing components may be excluded from the RNA molecules disclosed herein.

[0359]

H. Self - amplifying RNA (saRNA)

[0360] In some embodiments, the RNA molecule can be saRNA. "Self - amplifying RNA", "saRNA", and "replicon" refer to RNA that can self - replicate. Self - amplifying RNA molecules can be produced by using a replication component derived from, for example, an alphavirus and replacing the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. Self - amplifying RNA molecules are generally positive - strand molecules that can be directly translated after delivery to a cell, and this translation provides an RNA - dependent RNA polymerase, which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA causes the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as collinear sub - genomic transcripts, can themselves be translated to provide in - situ expression of the encoded gene of interest (e.g., viral antigen), and / or can be transcribed to provide other transcripts that are synonymous with the delivered RNA that is translated to provide in - situ expression of the antigen. The overall result of this series of transcriptions is the amplification of the number of introduced saRNA molecules, and thus the encoded gene of interest (e.g., viral antigen) becomes the major polypeptide product of the cell.

[0361] 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 can be excluded from the self - amplifying RNA molecules disclosed herein. In some embodiments, the self - amplifying RNA can also include 5′ - end and 3′ - end traction replication sequences, and optionally a heterologous sequence encoding a desired amino acid sequence (e.g., the antigen of interest). A sub - genomic promoter that directs the expression of the heterologous sequence can be included in the self - amplifying RNA. Optionally, the heterologous sequence (e.g., the antigen of interest) can be in - frame fused to other coding regions in the self - amplifying RNA and / or can be under the control of an internal ribosome entry site (IRES).

[0362] 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, such as including 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 can be excluded from the RNA molecules disclosed herein.

[0363] In some embodiments, the self - amplifying RNA molecule can have two open reading frames. The first (5') open reading frame can encode a replicase; the second (3') open reading frame can encode a polypeptide comprising the antigen of interest. In some embodiments, the RNA can have additional (e.g., downstream) open reading frames, for example to encode other antigens or to encode accessory polypeptides.

[0364] 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.

[0365] In some embodiments, the self - amplifying RNA molecule can encode a single polypeptide antigen, or optionally two or more polypeptide antigens, which are linked together (e.g., in tandem) in such a way that each of the sequences retains its identity when expressed in amino acid sequence form. The polypeptides produced from the self - amplifying RNA can then be produced in the form of fusion polypeptides or engineered in such a way that separate polypeptides or peptide sequences are produced.

[0366] In some embodiments, the self - amplifying RNAs described herein can encode one or more polypeptide antigens comprising a series of epitopes. In some embodiments, the self - amplifying RNAs described herein can encode epitopes capable of eliciting a helper T - cell response or a cytotoxic T - cell response or both.

[0367] In one embodiment, the self - amplifying RNA disclosed herein comprises a sub - genomic promoter having a sequence with at least, at most, exactly, or between any two of the following identities to SEQ ID NO: 54: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one embodiment, the sub - genomic promoter comprises the sequence of SEQ ID NO: 54.

[0368]

SEQ ID NO: 54 (RNA)

[0369] CCUGAAUGGA CUACGACAUA GUCUAGUCCG CCAAG

[0370] 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, for example comprising any one of alphavirus proteins nsP1, nsP2, nsP3, nsP4 and any combination thereof.

[0371] In one embodiment, the self-amplifying RNA disclosed herein comprises an alphavirus replicase, such as any one of or any combination of alphavirus proteins nsP1, nsP2, nsP3, and nsP4, which comprises a sequence having at least, at most, exactly, or between any two of the following identities with SEQ ID NOs: 55-58: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one embodiment, the alphavirus proteins nsP1, nsP2, nsP3, and nsP4 each separately comprise the sequences of SEQ ID NOs: 55-58.

[0372]

SEQ ID NO:55(nsP1 RNA)

[0373]

[0374]

SEQ ID NO:56(NSP2 RNA)

[0375]

[0376]

[0377]

SEQ ID NO:57(NSP3 RNA)

[0378]

[0379]

SEQ ID NO:58(NSP4 RNA)

[0380]

[0381]

[0382]

IV.RNA Transcription

[0383] In some embodiments, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0384] 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 cell-free 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, for example, 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. ed.), New York, N.Y. Humana Press, 2010; Brunelle, J. L. and Green, R., 2013, Chapter 5 - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology 530: 101-114; all of which are incorporated herein by reference).

[0385] Cloning vectors can be applied to generate transcripts. These cloning vectors are generally referred to as transcription vectors and are covered 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 appropriate 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., General protocols 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 PAGE in Recombinant and in vitro RNA syntheses, Methods 941 Conn G.L. (ed.), New York, N.Y. Humana Press, 2012, each of which is incorporated herein by reference). The promoter used to control transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, the in vitro transcription according to the present invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning nucleic acids (especially cDNA) and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0386] The synthetic IVT RNA product can be in vitro translated or directly introduced into cells, where the product can be translated. For RNA, the terms "expression" or "translation" refer to the process in which the mRNA strand guides the assembly of an amino acid sequence in the ribosome of a cell to produce a peptide or protein. 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, small nuclear RNA molecules, small nucleolar RNA molecules, etc. In some embodiments, 1, 2, 3, 4, 5 or more of the foregoing synthetic RNA products can be excluded. The IVT reaction generally utilizes a DNA template (e.g., a linear DNA template) as described herein and / or utilized, ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase.

[0387] In some embodiments, the mRNA is manufactured by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides. In some embodiments, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter used to control transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid (especially cDNA) and introducing it into an appropriate vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0388] In some embodiments, the starting materials for IVT can include a linearized DNA template, nucleotides, an RNase inhibitor, pyrophosphatase, and / or a polymerase (such as T7 RNA polymerase). The nucleotides can be manufactured in-house, obtained from a vendor, or synthesized. The 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 such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase; and / or mutant polymerases such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides (including chemically modified nucleic acids and / or nucleotides). In some embodiments, 1, 2, 3, 4, 5, or more of the aforementioned RNA polymerases can be excluded. Some examples exclude the use of DNase.

[0389] In some embodiments, the IVT process is carried out in a bioreactor. The bioreactor can include a mixer. In some embodiments, the nucleotides can be added to the bioreactor throughout the IVT process.

[0390] In some embodiments, in a bioreactor, after the IVT process, one or more post-IVT agents are added to the IVT mixture containing RNA. Exemplary post-IVT agents can 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 IVT, the post-IVT agent is incubated with the mixture in the bioreactor. In some embodiments, the bioreactor can contain at least, at most, exactly, or any two of the following (inclusive or exclusive) of the 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 that is or is not at least, at most, exactly, or any two of the following (inclusive or exclusive): 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 100 mg / mL RNA or more.

[0391] In some embodiments, the IVT mixture can include residual spermidine, residual DNA, residual protein, peptides, HEPES, EDTA, ammonium sulfate, cations (such as Mg 2+ , Na + , Ca 2+ ), RNA fragments, residual nucleotides, free phosphates, or any combination thereof. In some embodiments, 1, 2, 3, 4, 5, or more of the foregoing can be excluded from the IVT mixture.

[0392] The isolation and / or purification of the nucleic acids described herein can include, but is 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 clearance, quality assurance, and quality control. Additional non-limiting examples of purification procedures include Beads (Beckman Coulter Genomics, Danvers, MA), poly T beads, LNA™ oligomeric T capture probes, Inc, 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 Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). In some embodiments, one, two, three, four, five, or more of the foregoing purifications can be excluded.

[0393] When used with respect to nucleic acids, the term "purified", such as "purified nucleic acid", refers to a nucleic acid that is separated from at least one contaminant. A "contaminant" is any substance that renders another substance inappropriate, impure, or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) exists in a form or setting different from that in which it is found in nature, or in a form or setting different from that which existed prior to the processes and / or purification methods to which it was subjected.

[0394] 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 the buffer solution of at least a portion of the IVT mixture to produce a concentrated RNA solution as the retentate.

[0395] In some embodiments, both "ultrafiltration" and "diafiltration" refer to membrane filtration processes. Ultrafiltration typically uses membranes with pore sizes of at least, at most, exactly below, or between any two of the following (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 typically classified by molecular weight cut-off (MWCO) rather than pore size. For example, the MWCO can be at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa, and 10000 kDa. Those skilled in the art will understand that the filtration membrane can comprise different suitable materials, including, for example, polymers, cellulose, ceramics, etc., depending on the application. In some embodiments, membrane filtration may be more suitable for large-scale purification processes.

[0396] In some embodiments, ultrafiltration and diafiltration of the IVT mixture for purifying RNA can include: (1) direct flow filtration (DFF), also known as "dead-end" filtration, which applies a feed flow 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 flow filtration, in which the feed flow is passed parallel to the membrane surface, where a portion passes through the membrane (permeate), while the remainder (retentate) is retained and / or recycled back to the feed tank.

[0397] In some embodiments, filtration of the IVT mixture is carried out via TFF, which comprises an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some embodiments, the first diafiltration step is carried out in the presence of ammonium sulfate. The first diafiltration step can be configured to remove most of the impurities from the IVT mixture. In some embodiments, the second diafiltration step is carried out in the absence of ammonium sulfate. The second diafiltration step can be configured to transfer the RNA into a DS buffer formulation.

[0398] A filtration membrane with an appropriate MWCO can be selected for ultrafiltration in the TFF process. The MWCO of the TFF membrane determines which solutes can pass through the membrane into the filtrate and which solutes are retained in the retentate. The MWCO of the TFF membrane can be selected such that substantially all of the solutes of interest (e.g., the desired synthetic RNA species) are retained in the retentate, while undesired 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) are passed into the filtrate. In some embodiments, the retentate containing the desired synthetic RNA species can be recycled to the feed reservoir for re-filtration in additional cycles. In some embodiments, the MWCO of the TFF membrane can be at least, at most, exactly, or between any two of the following (inclusive or exclusive): 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or more. In some embodiments, the MWCO of the TFF membrane can be at least, at most, exactly, or between any two of the following (inclusive or exclusive): 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or more. In some embodiments, the MWCO of the TFF membrane can be or be about 250 - 350 kDa. In some embodiments, the MWCO of the TFF membrane (e.g., a cellulose-based membrane) can be or be about 30 - 300 kDa; 50 - 300 kDa, 100 - 300 kDa, or 200 - 300 kDa.

[0399] Diafiltration can be carried out discontinuously or continuously. For example, in continuous diafiltration, the diafiltration solution can 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 (such as salts, solvents, etc.) that can freely permeate through the membrane are removed. Using solvent removal as an example, each additional diafiltration 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 (such as salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) further reduces the concentration of small molecules (such as solvents). Continuous diafiltration generally requires a minimum volume for a given reduction of the molecule to be filtered. On the other hand, discontinuous diafiltration allows for rapid changes in the retentate state (such as pH, salt content, etc.). In some embodiments, the first diafiltration step is carried out in an amount of at least, at most, exactly below or between any two of the following (inclusive or exclusive): 2, 3, 4, 5, 6, 7, 8, 9, 10 or more diafiltration volumes. In some embodiments, the second diafiltration step is carried out in an amount of at least, at most, exactly below or between any two of the following (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 carried out with 5 diafiltration volumes and the second diafiltration step is carried out with 10 diafiltration volumes.

[0400] In some embodiments, for ultrafiltration and / or diafiltration, the IVT mixture is filtered at a rate of at least, at most, exactly below or between any two of the following (inclusive or exclusive): 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 1000 L / m 2 Filter area / hour or greater. The concentrated RNA solution can contain single-stranded RNA in an amount of at least, at most, exactly below or between any two of the following (inclusive or exclusive): 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5 mg / mL.

[0401] In some embodiments, the bioburden of the concentrated RNA solution obtained by filtering the RNA product solution can also be reduced. One or more filters can be used for filtering to reduce the bioburden. The one or more filters may include a filter having a pore size of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.

[0402] As an example, reducing the bioburden may include draining a retentate sump containing a retentate obtained from ultrafiltration and / or diafiltration to obtain a retentate. Reducing the bioburden may include flushing a filtration system for ultrafiltration and / or diafiltration with a wash buffer solution to obtain a wash tank solution containing residual RNA remaining in the filtration system. The retentate may be filtered to obtain a filtered retentate. The wash tank solution may be filtered using a first 0.2 μm filter to obtain a filtered wash tank solution. The retentate may be filtered using a first 0.2 μm filter or another 0.2 μm filter.

[0403] The filtered wash pool solution and the filtered retentate can be combined to form a combined pool 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.

[0404] 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.

[0405] In some embodiments, nucleic acids can be sequenced by methods including, but not limited to, reverse transcriptase-PCR.

[0406] In some embodiments, nucleic acids can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV / Vis). Non-limiting examples of UV / Vis spectrometers are Spectrometer (ThermoFisher, Waltham, MA). Quantified nucleic acids can be analyzed to determine whether the nucleic acid can be of 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 aforementioned assessment methods can be excluded.

[0407]

V.RNA Encapsulation

[0408] 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 lipids, lipid nanoparticles (LNPs), lipid complexes, polymer particles, polymeric complexes, integral delivery systems, or combinations thereof. In some embodiments, one, two, three, four, five or more of the foregoing components can be excluded as encapsulating agents.

[0409] In one embodiment, the encapsulating agent is a lipid, and RNA encapsulated in lipid nanoparticles (LNPs) is produced. Without being bound by any theory, it is believed that cationic or cationizable lipids or lipid-like materials and / or cationic polymers combine with nucleic acids to form aggregates, and this aggregation produces colloidally stable particles.

[0410] The lipid can be a naturally occurring lipid or a synthetic lipid. However, lipids are generally biomaterials. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfoglycolipids, lipids with ether and ester linkages of fatty acids, and polymerizable lipids, and combinations thereof. Lipids are substances insoluble in water and extractable with organic solvents. Compounds other than those specifically described herein are understood by those skilled in the art as lipids and are encompassed by the compositions and methods of the present invention. Lipid components and non-lipids can be covalently or non-covalently linked to each other.

[0411] In some embodiments, the LNPs can be designed to protect RNA molecules (such as saRNA, mRNA) from extracellular RNases, and / or can be engineered for systemic delivery of RNA to target cells. In some embodiments, such LNPs can be particularly suitable for delivering RNA molecules (such as saRNA, mRNA) when the RNA molecules are administered intravenously to an individual in need. In some embodiments, such LNPs can be particularly suitable for delivering RNA molecules (such as saRNA, mRNA) when the RNA molecules are administered intramuscularly to an individual in need. In some embodiments, such LNPs can be particularly suitable for delivering RNA molecules (such as saRNA, mRNA) when the RNA molecules are administered intradermally to an individual in need. In some embodiments, such LNPs can be particularly suitable for delivering RNA molecules (such as saRNA, mRNA) when the RNA molecules are administered intranasally to an individual in need.

[0412] In one embodiment, the concentration of RNA in the RNA product solution is < 1 mg / mL. In another embodiment, the concentration of RNA is at least or at least about 0.05 mg / mL. In another embodiment, the concentration of RNA is at least or at least about 0.5 mg / mL. In another embodiment, the concentration of RNA is at least or at least about 1 mg / ml. In another embodiment, the RNA concentration is or is about 0.05 mg / mL to about 0.5 mg / mL. In another embodiment, the concentration of RNA is at least 10 mg / mL. In another embodiment, the concentration of RNA is at least 50 mg / mL. In some embodiments, the concentration of RNA is or is not at least, at most, exactly below, between any two of the following (inclusive or exclusive) or about below: 0.05 mg / mL, 0.5 mg / mL, 1 mg / mL, 10 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / mL, 150 mg / mL, 200 mg / mL, 250 mg / mL, 300 mg / mL, 400 mg / mL or more.

[0413] The present invention provides an RNA product solution and a lipid formulation mixture or composition thereof, which comprises at least one RNA encoding, for example, an antigen (such as the prefusion F protein of RSV), the at least one RNA being 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.

[0414] Lipid nanoparticles or LNPs refer to any form of particles produced when a cationic lipid and optionally one or more other lipids are combined, for example, in an aqueous environment and / or in the presence of RNA. In some embodiments, the lipid nanoparticles are 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 (such as a cell, tissue, organ, tumor, etc.). In some embodiments, the lipid nanoparticles of the present invention contain nucleic acids (e.g., mRNA). Such lipid nanoparticles generally contain a cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, polymer-conjugated lipids or combinations thereof. In some embodiments, the LNP contains at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., steroid) and / or at least one polymer-conjugated lipid (e.g., polyethylene glycol (PEG)-modified lipid). In some embodiments, one, two, three or more of the foregoing excipients can be excluded from the LNP.

[0415] In some embodiments, the LNP comprises 20-60 mol% of one or more cationic (e.g., ionizable) lipids. For example, the LNP may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% of one or more cationic (e.g., ionizable) lipids. In some embodiments, the LNP comprises or does not comprise at least, at most, exactly, or any two of the following (inclusive or exclusive) of one or more cationic (e.g., ionizable) lipids: 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol%. In some embodiments, the LNP comprises 45-55 mole percent (mol%) of one or more cationic (e.g., ionizable) lipids. For example, the LNP may comprise or does not comprise at least, at most, exactly, or any two of the following (inclusive or exclusive) of one or more cationic (e.g., ionizable) lipids: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol%.

[0416] In some embodiments, the LNP comprises 5-25 mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% of one or more neutral (e.g., non-cationic) lipids. In some embodiments, the LNP is or is not at least, at most, exactly, or any two of the following (inclusive or exclusive) of one or more neutral (e.g., non-cationic) lipids: 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol%. In some embodiments, the LNP comprises 5-15 mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise at least, at most, exactly, or 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 15 mol%.

[0417] In some embodiments, the LNP comprises 25 to 55 mol% of one or more structural lipids (such as steroids). For example, the LNP may comprise 25 to 50 mol%, 25 to 45 mol%, 25 to 40 mol%, 25 to 35 mol%, 25 to 30 mol%, 30 to 55 mol%, 30 to 50 mol%, 30 to 45 mol%, 30 to 40 mol%, 30 to 35 mol%, 35 to 55 mol%, 35 to 50 mol%, 35 to 45 mol%, 35 to 40 mol%, 40 to 55 mol%, 40 to 50 mol%, 40 to 45 mol%, 45 to 55 mol%, 45 to 50 mol% or 50 to 55 mol% of one or more structural lipids (such as steroids). In some embodiments, the LNP is or is not at least, at most, exactly the following or any two of the following (inclusive or exclusive) of one or more structural lipids (such as steroids): 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol% or 55 mol%. In some embodiments, the LNP comprises 35 to 40 mol% of one or more structural lipids (such as steroids). For example, the LNP may comprise at least, at most, exactly the following or any two of the following (inclusive or exclusive) of one or more structural lipids (such as steroids): 35, 36, 37, 38, 39 or 40 mol%.

[0418] In some embodiments, the LNP comprises 0.5 to 15 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). For example, the lipid nanoparticle may comprise 0.5 to 10 mol%, 0.5 to 5 mol%, 1 to 15 mol%, 1 to 10 mol%, 1 to 5 mol%, 2 to 15 mol%, 2 to 10 mol%, 2 to 5 mol%, 5 to 15 mol%, 5 to 10 mol%, or 10 to 15 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). In some embodiments, the lipid LNP is or is not at least, at most, exactly the following, or any two of the following (inclusive or exclusive): 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). In some embodiments, the LNP comprises 1 to 2 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids). For example, the LNP may comprise at least, at most, exactly the following, or any two of the following (inclusive or exclusive): 1, 1.5, or 2 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids).

[0419] In some embodiments, the LNP comprises: 20 to 75 mol% of one or more cationic (e.g., ionizable) lipids (e.g., at least, at most, exactly, 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 mol% of one or more neutral (e.g., non-cationic) lipids (e.g., at least, at most, exactly, or between the following (inclusive or exclusive): 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 mol% of one or more structural lipids (e.g., steroids) (e.g., at least, at most, exactly, or between the following (inclusive or exclusive): 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%), and 0.5 to 20 mol% of one or more polymer-conjugated lipids (e.g., polyethylene glycol (PEG)-modified lipids) (e.g., at least, at most, exactly, or between the following (inclusive or exclusive): 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some embodiments, one, two, three, or more of the lipids may be excluded from the LNP.

[0420] In some non-limiting embodiments, the molar lipid ratio is 50 / 10 / 38.5 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 60 / 7.5 / 31 / 1.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 57.5 / 7.5 / 31.5 / 3.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 57.2 / 7.1 / 34.3 / 1.4 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 40 / 15 / 40 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 50 / 10 / 35 / 4.5 / 0.5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 50 / 10 / 35 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 40 / 10 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), 35 / 15 / 40 / 10 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid), or 52 / 13 / 30 / 5 (mol% cationic lipid / neutral lipid / structural lipid / polymer-conjugated lipid).

[0421] In some embodiments, an active agent or therapeutic agent (such as a nucleic acid (e.g., mRNA)) can be encapsulated in the lipid portion of the lipid nanoparticle and / or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesired effects induced by the mechanisms of the host organism or cell, such as an adverse immune response. The nucleic acid (e.g., mRNA) or a portion thereof can also be bound and complexed with the lipid nanoparticle. The lipid nanoparticle can comprise any lipid capable of forming a particle that links the nucleic acid and / or encapsulates one or more nucleic acids.

[0422] In some embodiments, the provided RNA molecules (e.g., saRNA, mRNA) can be formulated with LNPs. In some embodiments, the lipid nanoparticles can have or can not have an average diameter that is or is not about 1 to 500 nm (e.g., at least, at most, exactly below, or between (inclusive or exclusive) the following: 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 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or is at least, at most, exactly below, or between (inclusive or exclusive) the following: 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and is substantially non-toxic. The term "average diameter" refers to the average hydrodynamic diameter of the particles measured by dynamic light scattering (DLS) and data analysis using the so-called cumulant algorithm, the result of which provides the so-called Z-average of the length dimension and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter", or "size" of the particles is used synonymously with the Z-average.

[0423] The LNPs described herein can exhibit a polydispersity index less than or less than about 0.5, 0.4, 0.3, or 0.2 or smaller. By way of example, the LNPs can exhibit or not exhibit a polydispersity index that is at least, at most, exactly below, or between (inclusive or exclusive) the following: 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. In some embodiments, the polydispersity index is calculated by so-called cumulant analysis as referred to in the definition of "average diameter" based on dynamic light scattering measurements. Under certain preconditions, it can be regarded as a measure of the size distribution of the nanoparticle population.

[0424] In some embodiments, the LNPs of the present invention contain or do not contain an N:P ratio that is or is about 2:1 to about 30:1, such as an N:P ratio that is at least, at most, exactly below, or between (inclusive or exclusive) the following: 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. In some embodiments, the LNPs of the present invention contain an N:P ratio that is or is about 6:1. In some embodiments, the LNPs of the present invention contain an N:P ratio that is or is about 3:1.

[0425] In some embodiments, the LNP of the present invention contains or does not contain a cationic lipid component to RNA wt / wt ratio that is or is about 5:1 to about 100:1, such as at least, at most, exactly below, or between (inclusive or exclusive) the following cationic lipid component to RNA wt / wt ratios: 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. In some embodiments, the LNP of the present invention contains an ionizable cationic lipid component to RNA wt / wt ratio that is or is about 20:1. In some embodiments, the LNP of the present invention contains an ionizable cationic lipid component to RNA wt / wt ratio that is or is about 10:1.

[0426] In certain embodiments, the nucleic acid (e.g., an RNA molecule) (when present in the provided LNP) is resistant to nuclease degradation in an aqueous solution. In some embodiments, the LNP is a liver-targeting lipid nanoparticle. In some embodiments, the LNP is a cationic lipid nanoparticle comprising one or more cationic lipids (such as those described herein). In some embodiments, the cationic LNP can comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (such as at least one neutral lipid).

[0427] In certain embodiments, the RNA solution and its lipid formulation mixture or composition can have at least, at most, exactly the following, between the following (inclusive or exclusive) or about the following specific lipids, lipid types or non-lipid components (such as lipid-like materials and / or cationic polymers and / or adjuvants), antigens, peptides, polypeptides, sugars, nucleic acids or other materials disclosed herein or known to those skilled in the art: 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%.

[0428] The LNPs described herein can be produced using components, compositions and methods generally known in the art, see for example 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 / US...

Claims

1. An RNA molecule comprising at least one open reading frame encoding a respiratory syncytial virus (RSV) fusion protein F (F) polypeptide.

2. The RNA molecule according to claim 1, wherein the RSV polypeptide is a full-length polypeptide, a truncated polypeptide, a fragment thereof, or a variant.

3. The 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 to 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 having at least 90%, 95%, 96%, 97%, 98%, or 99% identity 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 having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of the sequences of SEQ ID NOs: 11-16 and 63-70.

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: 11-16 and 63-70.

8. The RNA molecule according to claim 1, which further comprises 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: 17-19.

10. The RNA molecule according to claim 1, which further comprises 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: 20-25.

12. The RNA molecule according to claim 1, wherein the RNA molecule further comprises a 5' cap portion or a 3' polyA tail.

13. The RNA molecule according to claim 12, wherein the polyA tail comprises the sequence having SEQ ID NO:

26.

14. The RNA molecule according to claim 1, wherein the open reading frame has a G / C content of at least 55%, 60%, 65%, 70%, or 75%, or is or about 50%-75% or 55%-70%.

15. The RNA molecule according to claim 1, wherein the encoded RSV polypeptide is localized in the cell membrane, localized in the Golgi, and / or 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-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-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 the 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 nanoparticle comprises at least one of the following: a cationic lipid, a polyethylene glycolated lipid, a neutral lipid, and a steroid or steroid analog.

23. The composition according to claim 22, wherein the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

24. A composition according to claim 22, wherein the polyethylene glycolated lipid is PEG-modified phosphatidylethanolamine; PEG-modified phosphatidic acid; PEG-modified ceramide (such as PEG-CerC14 or PEG-CerC20); PEG-modified dialkylamine; PEG-modified diacylglycerol; PEG-modified dialkylglycerol; 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide; diol 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; polyethylene glycolated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); polyethylene glycolated phosphatidylethanolamine (PEG-PE); PEG succinoyl diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl)-1-O-((ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); polyethylene glycolated ceramide (PEG-cer); or PEG dialkoxypropyl carbamate, 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.

25. A composition according to claim 24, wherein the polyethylene glycolated lipid is 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide (ALC-0159).

26. A composition according to claim 22, wherein the neutral lipid is distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE) and 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-dielaidoyl-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 the RNA molecule according to claim 1.

30. A method of preventing, treating or ameliorating an RSV-related infection, disease or condition in an individual, comprising administering to the individual an effective amount of the 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 age of the individual is less than about 1 year, about 1 year or older, about 5 years or older, about 10 years or older, about 20 years or older, about 30 years or older, about 40 years or older, about 50 years or older, about 60 years or older, about 70 years or older, or older.

33. The 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 individual is administered a single dose, two doses, three doses or more doses of the RNA molecule and, optionally, a booster dose 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 the composition according to claim 22.

36. A method of preventing, treating or ameliorating an RSV-related infection, disease or condition in an individual, comprising administering to the individual an effective amount of the composition according to claim 22.

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