JP2024151316A - RNA molecule - Google Patents

Abstract

The present invention provides compositions for preventing, treating or ameliorating infections, diseases or conditions associated with Varicella-Zoster Virus (VZV).
The present disclosure relates to RNA molecules encoding Varicella Zoster Virus (VZV). The present disclosure further relates to compositions comprising the RNA molecules formulated in lipid nanoparticles (RNA-LNPs). The present disclosure further relates to the use of the RNA molecules, RNA-LNPs and compositions for the treatment or prevention of herpes zoster or shingles.
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Description

2. Background Varicella zoster virus (VZV), also known as human herpesvirus 3 (HHV-3), is a human pathogen that causes chickenpox or chickenpox in children and later re-emerges as herpes zoster or shingles. Two vaccines have been developed and licensed in various countries to prevent herpes zoster. The first is ZOSTAVAX® (Merck & Co., Inc., Kenilworth, NJ, USA), a live attenuated VZV vaccine. The US FDA approved ZOSTAVAX® in 2006, but as of November 2020, ZOSTAVAX® is no longer available in the US. The second is SHINGRIX® (GlaxoSmithKline, Rockville, MD, USA), an AS01 _B- adjuvanted VZV gE subunit protein vaccine. The US FDA approved SHINGRIX® in 2017.

In the United States, approximately 1 million cases of shingles occur annually, and approximately 30% of all people infected with chickenpox later develop shingles. The incidence of shingles continues to increase worldwide. Therefore, given the high prevalence of shingles, there remains a need for improved vaccines for the prevention of shingles.

The present disclosure provides immunogenic compositions and methods for treating a subject comprising administration of an RNA molecule, e.g., an immunogenic RNA polynucleotide, encoding an amino acid sequence, e.g., an immunogenic antigen, including a Varicella-Zoster Virus (VZV) protein, an immunogenic variant thereof, or an immunogenic fragment of a VZV protein or an immunogenic variant thereof, e.g., an antigenic peptide or protein. Thus, the immunogenic antigen comprises an epitope of a VZV protein for inducing an immune response against VZV in a subject. The RNA polynucleotide encoding the immunogenic antigen is administered to provide an antigen (after expression of the polynucleotide by an appropriate target cell) for induction, e.g., stimulation, priming, and/or expansion of an immune response, e.g., antibodies and/or immune effector cells. In one aspect, the immune response induced according to the present disclosure is a B cell-mediated immune response, e.g., an antibody-mediated immune response. Additionally or alternatively, the immune response induced according to the present disclosure may be a T cell-mediated immune response. In one embodiment, the immune response is an anti-VZV immune response.

The immunogenic compositions described herein include (as active ingredients) RNA molecules that contain RNA that can be translated into protein in the recipient's cells. In addition to a wild-type or codon-optimized sequence encoding an antigen sequence, the RNA molecule may contain one or more structural elements (5' cap, 5' UTR, 3' UTR, poly-A-tail) that are optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency. In one embodiment, the RNA molecule contains all of these elements. In some embodiments, each uridine of the RNA molecule is replaced by N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA). The RNA molecules described herein may be formulated with lipids and/or proteins to generate RNA-particles (e.g., lipid nanoparticles (LNPs)) for administration, encapsulated in lipids and/or proteins, or complexed with lipids and/or proteins. In one embodiment, the RNA molecules described herein are formulated with lipids, encapsulated in lipids, or complexed with lipids to produce RNA-lipid nanoparticles (e.g., RNA-LNPs) for administration. In one embodiment, the RNA molecules described herein are formulated with proteins, encapsulated in proteins, or complexed with proteins for administration. In one embodiment, the RNA molecules described herein are formulated with lipids and proteins, encapsulated in lipids and proteins, or complexed with lipids and proteins for administration. When a combination of different RNA molecules is used, the RNA molecules may be formulated together with lipids and/or proteins to produce RNA-particles for administration, or may be formulated separately from lipids and/or proteins.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one open reading frame (ORF) encoding a VZV antigen. In some embodiments, the VZV antigen is a VZV polypeptide. In some embodiments, the VZV polypeptide is a VZV glycoprotein. In some embodiments, the VZV glycoprotein is VZV gK, gN, gC, gB, gH, gM, gL, gI, or gE. In some embodiments, the VZV glycoprotein is VZV gE. In some embodiments, the VZV polypeptide is full-length, truncated, fragment, or variant thereof. In some embodiments, the VZV polypeptide comprises at least one mutation.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF encoding a VZV polypeptide of Table 1. In some embodiments, the VZV polypeptide has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher percent identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher percent identity, or up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher percent identity, to any of the amino acid sequences of Table 1, e.g., any of SEQ ID NOs: 1-11. In some embodiments, the VZV polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-11. In some embodiments, the VZV polypeptide consists of any of the amino acid sequences in Table 1, e.g., any of SEQ ID NOs: 1-11.

The present disclosure provides RNA molecules and RNA-LNPs comprising at least one ORF transcribed from at least one DNA nucleic acid of Table 2. In some embodiments, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, or up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, to any of the nucleic acid sequences of Table 2, e.g., any of SEQ ID NOs: 12-145. In some embodiments, the RNA molecule is transcribed from a nucleic acid sequence selected from SEQ ID NOs: 12-145. In some embodiments, the RNA molecule comprises an ORF transcribed from any of the nucleic acid sequences in Table 2, e.g., any of SEQ ID NOs: 12-145.

The present disclosure further provides RNA molecules and RNA-LNPs comprising at least one ORF comprising an RNA nucleic acid sequence of Table 3. In some embodiments, the RNA molecule comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, or up to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher percent identity, to any of the nucleic acid sequences of Table 3, e.g., any of SEQ ID NOs: 146-279. In some embodiments, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 146-279. In some embodiments, the RNA molecule comprises a nucleic acid sequence consisting of any of the nucleic acid sequences in Table 3, e.g., any of SEQ ID NOs: 146-279. In some embodiments, each uridine in any of SEQ ID NOs: 146-279 is replaced by N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure further provides RNA molecules and RNA-LNPs comprising a 5' untranslated region (5'-UTR) and/or a 3' untranslated region (3'-UTR). In some embodiments, the RNA molecule comprises a 5' untranslated region (5'-UTR). In some embodiments, the 5'UTR comprises a sequence selected from any of SEQ ID NOs: 281 (SEQ ID NO: 280 - DNA; SEQ ID NO: 282 - RNA with Ψ) and 312-313. 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 percent identity to any of SEQ ID NOs: 281 and 312-313. In some embodiments, the 5'UTR comprises a sequence selected from any of SEQ ID NOs: 281 and 312-313. In some embodiments, the 5'UTR comprises a sequence consisting of any of SEQ ID NOs: 281 and 312-313.

In some embodiments, the RNA molecules and RNA-LNPs comprise a 3' untranslated region (3'-UTR). In some embodiments, the 3'UTR comprises a sequence selected from any of SEQ ID NOs: 284 (SEQ ID NO: 283 - DNA; SEQ ID NO: 285 - RNA with Ψ), 314 and 317 (SEQ ID NO: 318 - RNA with Ψ). 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 percent identity to any of SEQ ID NOs: 284, 314 and 317. In some embodiments, the 3'UTR comprises a sequence selected from any of SEQ ID NOs: 284, 314 and 317. In some embodiments, the 3'UTR comprises a sequence consisting of any of SEQ ID NOs: 284, 314 and 317.

The present disclosure further provides RNA molecules and RNA-LNPs comprising a 5' cap portion. In some embodiments, the 5' cap portion is (3'OMe) _-m27,3' ^-O Gppp( _m12' ^-O )ApG. The present disclosure further provides RNA molecules and RNA-LNPs comprising a 3' poly-A tail. In some embodiments, the poly-A tail comprises a sequence selected from any of SEQ ID NOs:287 (SEQ ID NO:286 - DNA; SEQ ID NO:288 - RNA having Ψ) and 315 (SEQ ID NO:316 - RNA having Ψ). In some embodiments, the poly-A tail comprises a sequence selected from any of SEQ ID NOs:287 and 315 +/- 1 adenosine (A) or +/- 2 adenosines (A).

In some embodiments, the RNA molecule comprises a 5'UTR and a 3'UTR. In some embodiments, the RNA molecule comprises a 5' cap, a 5'UTR, and a 3'UTR. In some embodiments, the RNA molecule comprises a 5' cap, a 5'UTR, a 3'UTR, and a polyA tail. In some embodiments, the RNA molecule comprises a 5'UTR, a 3'UTR, and a polyA tail. In some embodiments, each uridine in any of the 5'UTR, 3'UTR, and polyA tail is replaced by N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure provides RNA molecules as set forth in Table 5. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:146, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (gE WT). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:147, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (gE WT CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:148, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (gE WT CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:149, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms3 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:150, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms3 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:151, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms4 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:152, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms4 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:153, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms5 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:154, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms5 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:155, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms5 CO2 v2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:156, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms6 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:157, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms6 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:158, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms8 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:159, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms9 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:160, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms9 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:161, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms10 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:162, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms10 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:163, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms10 CO3). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:164, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms11 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:165, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms11 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:166, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms12 CO1). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:167, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315 (ms12 CO2). In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:168, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:169, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:170, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:171, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:172, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:173, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:174, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of any one of SEQ ID NOs:175-238, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of SEQ ID NO:239, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of any one of SEQ ID NOs:240-254, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of any one of SEQ ID NOs:255-267, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the RNA molecule comprises a 5'UTR of SEQ ID NO:281 or 312, a VZV ORF of any one of SEQ ID NOs:268-279, a 3'UTR of SEQ ID NO:284 or 317, and/or a polyA tail of SEQ ID NO:287 or 315. In some embodiments, the VZV ORF further comprises a stop codon as described herein. In some embodiments, the length of the polyA tail may contain +1/-1 A or +2/-2 A. In some embodiments, each uridine of the RNA molecule is replaced by N1-methylpseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure further provides an RNA molecule comprising at least one open reading frame generated from codon-optimized DNA. In some embodiments, the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, about 50%-75%, or about 55%-70%. In some embodiments, the G/C content is about 58%, about 66%, or about 62%. The present disclosure further provides an RNA molecule encoding a VZV polypeptide that is localized in the cell membrane, localized in the Golgi, and/or anchored in the membrane and secreted. The present disclosure further provides an RNA molecule comprising a stabilized RNA. The present disclosure further provides an RNA molecule comprising 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).

The present disclosure further provides an RNA molecule that is messenger RNA (mRNA) or a self-replicating RNA. In some embodiments, the RNA is mRNA.

The present disclosure further provides immunogenic compositions comprising the RNA molecules described herein. The RNA molecules may be formulated in, encapsulated in, complexed with, bound to, or adsorbed to lipid nanoparticles (LNPs) (e.g., VZV RNA-LNPs) in such immunogenic compositions. In some embodiments, the lipid nanoparticles comprise at least one of a cationic lipid, a PEGylated lipid, and at least one structural lipid (e.g., a neutral lipid and a steroid or steroid analog).

In some embodiments, the lipid nanoparticles include a cationic lipid. In some embodiments, the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

In some embodiments, the lipid nanoparticles comprise a lipid conjugated to a polymer. In some embodiments, the lipid nanoparticles comprise a PEGylated lipid, also referred to as a PEG lipid. In some embodiments, the PEGylated lipid is a glycol lipid including 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-ditetradecylacetamide, PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyri[pi]oxypropyl]-1,2-diamine (PEG-DMG), or PEG-2000-DMG. Ristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

In some embodiments, the lipid nanoparticles include at least one structured lipid, such as a neutral lipid. In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl- Selected from phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and/or 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

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

In some embodiments, the lipid nanoparticles have an average diameter of about 1 to about 500 nm.

In some embodiments, the RNA-LNP immunogenic composition comprises an RNA-LNP having a lipid composition comprising at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to ... encapsulated in LNPs having a lipid composition comprising a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a first structural lipid at a concentration of about 0.1-0.25 mg/mL, and a second structural lipid at a concentration of about 0.3-0.45 mg/mL. A liquid RNA-LNP composition comprising an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein at a concentration of 30, 0.45, 0.60, 0.75, or 0.90 mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL. In some embodiments, the liquid composition further comprises a buffer composition comprising a first buffer at a concentration of about 0.15-0.3 mg/mL, a second buffer at a concentration of about 1.25-1.4 mg/mL, and a stabilizer at a concentration of about 95-110 mg/mL.

In a specific embodiment, the liquid RNA-LNP immunogenic composition is an LNP having a lipid composition comprising ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of about 0.8-0.95 mg/mL, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of about 0.05-0.15 mg/mL, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of about 0.1-0.25 mg/mL, and cholesterol at a concentration of about 0.3-0.45 mg/mL. The encapsulated RNA molecule/polynucleotide encoding a VZV polypeptide disclosed herein comprises a concentration of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL. In some embodiments, the liquid composition further comprises a Tris buffer composition comprising tromethamine at a concentration of about 0.1-0.3 mg/mL and Tris hydrochloride (HCl) at a concentration of about 1.25-1.4 mg/mL, and sucrose at a concentration of about 95-110 mg/mL.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, of RNA encapsulated in LNPs; or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, of an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, and further comprising about 5-15 mM Tris buffer, 200-400 mM sucrose at a pH of about 7.0-8.0.

In some embodiments, the RNA-LNP immunogenic composition comprises an RNA-LNP having a lipid composition comprising at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to ...75, or 0.90 mg/mL, encapsulated in LNPs having a lipid composition comprising a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a first structural lipid at a concentration of about 0.1-0.25 mg/mL, and a second structural lipid at a concentration of about 0.3-0.45 mg/mL. 0.01-0.09mg/mL。 Lyophilized (reconstituted) RNA-LNP composition comprising an RNA molecule / polynucleotide encoding a VZV polypeptide disclosed herein at a concentration of about 0.01-0.09mg/mL, 0.5, 0.60, 0.75, or 0.90mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably about 0.01-0.09mg/mL. In some embodiments, the lyophilized composition further comprises a first buffering agent at a concentration of about 0.01-0.15mg/mL, a second buffering agent at a concentration of about 0.5-0.65mg/mL, a stabilizer at a concentration of about 35-50mg/mL, and a salt diluent at a concentration of about 5-15mg/mL for reconstitution. In a specific embodiment, the lyophilized composition is reconstituted in about 0.6-0.75 mL of salt diluent. The concentration in the lyophilized RNA-LNP composition is determined after reconstitution.

In a specific embodiment, the lyophilized (reconstituted) RNA-LNP composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in LNPs having a lipid composition of ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, and cholesterol at a concentration of about 0.3-0.45 mg/mL. and a Tris buffer composition comprising tromethamine at a concentration of about 0.01-0.15 mg/mL and Tris HCl at a concentration of about 0.5-0.65 mg/mL, sucrose at a concentration of about 35-50 mg/mL, and a sodium chloride (NaCl) diluent at a concentration of about 5-15 mg/mL for reconstitution. In a specific embodiment, the lyophilized composition is reconstituted in about 0.6-0.75 mL of sodium chloride. The concentration in the lyophilized RNA-LNP composition is determined after reconstitution.

The present disclosure provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered to a subject at a dose of at least 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, up to 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, exactly 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or between any two or more of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg per administration of VZV RNA encapsulated in LNPs.

The present disclosure provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered in a single dose. The present disclosure further provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered twice (e.g., at day 0 and about day 7, day 0 and about day 14, day 0 and about day 21, day 0 and about day 28, day 0 and about day 60, day 0 and about day 90, day 0 and about day 120, day 0 and about day 150, day 0 and about day 180, day 0 and about 1 month later, day 0 and about 2 months later, day 0 and about 3 months later, day 0 and about 6 months later, day 0 and about 9 months later, day 0 and about 12 months later, day 0 and about 18 months later, day 0 and about 2 years later, day 0 and about 5 years later, or day 0 and about 10 years later). The present disclosure further provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered twice at day 0 and about 2 months later. The present disclosure further provides RNA molecules, RNA-LNPs and immunogenic compositions that may be administered twice, at day 0 and about 6 months later. The present disclosure further provides RNA molecules, RNA-LNPs and immunogenic compositions that may be administered in 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more doses. In some embodiments, periodic boosters at 1-5 year intervals may be desired to maintain protective levels of antibodies. The present disclosure further provides for administration of at least one booster dose.

The present disclosure provides a method of inducing an immune response to VZV in a subject, the method comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein. The present disclosure further provides for the use of an RNA molecule, RNA-LNP and/or immunogenic composition described herein in the production of a medicament for use in inducing an immune response to VZV in a subject.

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

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

The present disclosure provides a method of preventing, treating, or ameliorating an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule, RNA-LNP, and/or immunogenic composition described herein. The present disclosure further provides a use of an RNA molecule, RNA-LNP, and/or immunogenic composition described herein in the manufacture of a medicament for use in preventing, treating, or ameliorating an infection, disease, or condition in a subject. In some embodiments, the infection, disease, or condition is associated with VZV. In some embodiments, the infection, disease, or condition is herpes zoster (shingles). In some embodiments, the infection, disease, or condition is post-herpetic neuralgia.

The present disclosure provides a method of preventing, treating, or ameliorating an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule and/or RNA-LNP or immunogenic composition comprising at least one open reading frame encoding a VZV polypeptide described herein. The present disclosure further provides for the use of an RNA molecule and/or RNA-LNP or immunogenic composition comprising at least one open reading frame encoding a VZV polypeptide described herein in the manufacture of a medicament for use in preventing, treating, or ameliorating an infection, disease, or condition in a subject. In some embodiments, the infection, disease, or condition is associated with VZV. In some embodiments, the infection, disease, or condition is herpes zoster or shingles. In some embodiments, the infection, disease, or condition is post-herpetic neuralgia.

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

In some embodiments, the subject is, is at least, or is at most about 1 year old, 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. In some embodiments, the subject is about 50 years or older.

In some embodiments, the subject is immunocompetent. In some embodiments, the subject is immunocompromised.

The present disclosure 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 disclosure provides methods or uses described herein, wherein the RNA molecules, RNA-LNPs and/or immunogenic compositions are administered by intradermal or intramuscular injection.

It is contemplated that any aspect discussed herein may be implemented with respect to any method or composition of the present disclosure, and vice versa. Furthermore, a composition of the present disclosure may be used to achieve a method of the present disclosure.

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

Figure 1 shows a schematic of wild-type (WT) Varicella Zoster Virus (VZV) gE protein (gE WT) and variant VZV gE proteins, where SP refers to the signal peptide sequence, ectodomain refers to the peptide sequence corresponding to the portion of the protein that extends into the extracellular space, TM refers to the transmembrane peptide sequence corresponding to the portion of the protein that spans the cell membrane, and CT refers to the cytoplasmic tail peptide sequence corresponding to the portion of the protein that extends into the cytoplasm. The variant VZV gE proteins with cytoplasmic tail modifications are designated ms4, ms5, ms8, ms9, ms10, ms11, and ms12. The secreted variant VZV gE proteins with TM modifications are designated ms3 and ms6. A VZV gE RNA construct encoding the VZV gE protein was generated from codon-optimized (CO) DNA, where CO1 indicates a CO construct with a G/C content of about 58%, CO2 indicates a CO construct with a G/C content of about 66%, and CO3 indicates a CO construct with a G/C content of about 62%. Figure 2 shows VZV gE expression by Vero cells transfected with 10 ng, 25 ng, or 50 ng of VZV RNA constructs, including WT constructs (gE_WT CO1 and gE_WT CO2) and constructs with modifications in the cytoplasmic tail (CT) (ms4, ms5, ms8, ms9, ms10, ms11, and ms12; CO1 and CO2 for each). Cells were imaged for VZV gE expression, and the percentage of VZV gE ⁺ transfected Vero cells is shown for each VZV RNA construct. Figure 3 shows VZV gE expression by Vero cells transfected with 10 ng, 25 ng, or 50 ng of VZV RNA constructs (ms3 and ms6; CO1 and CO2 for each) with modifications in the (secreted) transmembrane (TM). Cells were imaged for VZV gE expression, and the percentage of VZV gE ⁺ transfected Vero cells is shown for each VZV RNA construct. FIG. 4 shows the mean fluorescence intensity (MFI) of Vero cells transfected with 10 ng, 25 ng, or 50 ng of VZV RNA constructs, including WT constructs (gE_WT CO1 and gE_WT CO2) and constructs with modifications in the cytoplasmic tail (CT) (ms4, ms5, ms8, ms9, ms10, ms11, and ms12; CO1 and CO2 for each). FIG. 5 shows the mean fluorescence intensity (MFI) of Vero cells transfected with 10 ng, 25 ng, or 50 ng of VZV RNA constructs (ms3 and ms6; CO1 and CO2 for each) with modifications in the (secreted) transmembrane (TM). FIG. 6 shows the subcellular localization of VZV gE at 63-fold magnification in Vero cells transfected with 50 ng of gE WT VZV RNA construct and variant VZV gE RNA constructs with cytoplasmic tail modifications (ms4, ms5, ms8). FIG. 7 shows the subcellular localization of VZV gE at 63-fold magnification in Vero cells transfected with 50 ng of variant VZV gE RNA constructs (ms9, ms11, and ms12) with cytoplasmic tail modifications. FIG. 8 shows the subcellular localization of VZV gE at 63-fold magnification in Vero cells transfected with 50 ng of a variant VZV gE RNA construct (ms10) with a cytoplasmic tail modification. FIG. 9 shows the subcellular localization of VZV gE at 63-fold magnification in Vero cells transfected with 50 ng of (secreted) variant VZV gE RNA constructs (ms3 and ms6) with TM modifications. FIG. 10 shows the subcellular localization of VZV gE at 10-fold magnification in Vero cells transfected with 25 ng of gE WT VZV RNA construct and variant VZV gE RNAs with cytoplasmic tail modifications (ms4, ms5, ms8). FIG. 11 shows the subcellular localization of VZV gE at 10-fold magnification in Vero cells transfected with 25 ng of variant VZV gE RNA constructs (ms9, ms11, ms12) with cytoplasmic tail modifications. FIG. 12 shows the subcellular localization of VZV gE at 10-fold magnification in Vero cells transfected with 25 ng of a variant VZV gE RNA construct (ms10) with a cytoplasmic tail modification. FIG. 13 shows the subcellular localization of VZV gE at 10-fold magnification in Vero cells transfected with 25 ng of (secreted) variant VZV gE RNA constructs (ms3 and ms6) with TM modifications. FIG. 14 shows titration curves generated by plotting the amount of input RNA versus the percentage of VZV gE-expressing human embryonic kidney (HEK) 293T cells, where the titration curves are used to determine the EC50 of VZV RNA-LNP vaccines formulated with VZV gE WT RNA constructs (gE WT CO1 and gE WT CO2), a variant VZV gE RNA construct with a cytoplasmic tail modification (ms4 CO1), and a variant VZV gE RNA construct with a (secreted) transmembrane (TM ₎ modification (ms3 CO1). FIG. 15 shows titration curves generated by plotting the amount of input RNA versus the percentage of VZV gE-expressing human embryonic kidney (HEK) 293T cells, where the titration curves are used to determine the EC50 of VZV RNA-LNP vaccines formulated with VZV gE WT RNA constructs (gE WT CO1 and gE WT CO2), a variant VZV gE RNA construct with a cytoplasmic tail modification (ms5 CO1), and a variant VZV gE RNA construct with a (secreted ₎ TM modification (ms6 CO2). FIG. 16 shows the results of 1 μg, 2.5 μg, or 5 μg of SHINGRIX®; 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms3 CO1 RNA construct with a (secreted) TM modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms4 CO1 RNA construct with a cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct (gE_WT CO1 1 shows serum IgG levels in mice on day 28 after immunization on day 0 with a priming dose of IgG1 (LYO). FIG. 17 shows the results of 1 μg, 2.5 μg, or 5 μg of SHINGRIX®; 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms3 CO1 RNA construct with a (secreted) TM modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms4 CO1 RNA construct with a cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct (gE_WT CO1 1 shows serum IgG levels in mice on day 34 after immunization with a priming dose of IgG1 and a booster dose of IgG1 and IgG2 (LYO) on day 0 and day 28, respectively. FIG. 18 shows the results of 1 μg, 2.5 μg, or 5 μg of SHINGRIX®; 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms3 CO1 RNA construct with a (secreted) TM modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a ms4 CO1 RNA construct with a cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine formulated with a gE_WT CO1 RNA construct (gE_WT CO1 1 shows serum IgG levels in mice on day 42 following immunization with a priming dose of IgG10 (LYO) on day 0 and a booster dose on day 28. FIG. 19 shows a comparison of serum IgG levels in mice on days 28 (priming dose only), 34 (6 days after the booster dose), and 42 (2 weeks after the booster dose) following immunization on day 0 with a priming dose of SHINGRIX® (1 μg) or VZV RNA-LNP vaccine (0.5 μg) and on day 28 with a booster dose. FIG. 20 shows the percentage of CD4+ IFN-γ+ staining T cells following ex vivo stimulation of splenocytes harvested from mice on day 34 following immunization on day 0 with a priming dose of SHINGRIX® (1 μg, 2.5 μg, or 5 μg) or VZV RNA ^- LNP vaccine (0.5 μg ^and /or 1 μg) and on day 28 with a booster dose. FIG. 21 shows the percentage of CD8+ IFN-γ+ staining T cells following ex vivo stimulation of splenocytes harvested from mice on day 34 following immunization on day 0 with a priming dose of SHINGRIX® (1 μg, 2.5 μg, or 5 μg) or VZV RNA ^- LNP vaccine (0.5 μg ^and /or 1 μg) and on day 28 with a booster dose. FIG. 22 shows serum IgG levels in mice 35 days after subcutaneous vaccination with 1350 pfu of live attenuated varicella (LAV) on day 0. FIG. 23 shows serum IgG levels in mice at day 63 (1 month after dose 1) following vaccination with the LAV vaccine on day 0 and immunization on day 35 with a dose of SHINGRIX® (5 μg, 2.5 μg, or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg or 0.5 μg); or VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a (secreted) TM modification (1 μg or 0.5 μg). FIG. 24 shows serum IgG levels in mice at day 76 (dose 2/13 days after boost) following vaccination with the LAV vaccine on day 0 and immunization on days 35 and 63 with doses of SHINGRIX® (5 μg, 2.5 μg, or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg or 0.5 μg); or VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a (secreted) TM modification (1 μg or 0.5 μg). FIG. 25 shows day 63 (1 month after dose 1) following vaccination with LAV vaccine on day 0 and immunization on day 35 with a dose of VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (1 μg or 0.5 μg) or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (gE_WT CO2 lyo) (1 μg or 0.5 μg); and day 63 (1 month after dose 1) following vaccination with LAV vaccine on day 0 and immunization on day 35 with a dose of VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (1 μg or 0.5 μg) or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (gE_WT CO2 lyo) (1 μg or 0.5 μg). 1 shows serum IgG levels in mice on day 76 (13 days after dose 2/boost) following immunization on days 35 and 63 with doses of IgG1 (lyo) (1 μg or 0.5 μg). FIG. 26A shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 48 (13 days after dose 1) in LAV-experienced mice immunized on day 35 with a dose of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 26A shows the number of IFN-γ-secreting VZV gE-specific cells as determined by ELISpot, and the results are expressed as spot-forming cells (SFC) per million cells. FIG. 26B shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 48 (13 days after dose 1) in LAV-experienced mice immunized on day 35 with a dose of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 26B shows IFN-γ expressing cells within CD4 ⁺ T cells measured by ICS assay, expressed as the percentage of IFN-γ ⁺ cells within CD4 ⁺ T cells. FIG. 26C shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 48 (13 days after dose 1) in LAV-experienced mice immunized on day 35 with a dose of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 26C shows IFN-γ expressing cells within CD8 + T cells measured by ICS assay, expressed as the percentage of IFN-γ ⁺ cells within CD8 ⁺ T cells. FIG. 26D shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 48 (13 days after dose 1) in LAV-experienced mice immunized on day 35 with a dose of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 26D shows B cell responses assessed in splenocytes by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry. FIG. 27A shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 76 (13 days after dose 2/boost) in LAV-experienced mice immunized on days 35 and 63 with doses of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 27A shows IFN-γ expressing cells within CD4 ⁺ T cells measured by ICS assay, expressed as the percentage of IFN-γ ⁺ cells within CD4 ⁺ T cells. FIG. 27B shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 76 (13 days after dose 2/boost) in LAV-experienced mice immunized on days 35 and 63 with doses of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 27B shows IFN-γ expressing cells within CD8 + T cells measured by ICS assay, expressed as the percentage of IFN-γ ⁺ cells within CD8 ⁺ T cells. FIG. 27C shows vaccine-induced VZV gE-specific T and B cell responses in splenocytes harvested on day 76 (13 days after dose 2/boost) in LAV-experienced mice immunized on days 35 and 63 with doses of SHINGRIX® (5 μg or 1 μg); VZV RNA-LNP vaccine formulated with a gE_WT CO2 RNA construct (1 μg or 0.5 μg); VZV RNA-LNP vaccine formulated with a ms5 CO1 RNA construct with a cytoplasmic tail modification (1 μg); VZV RNA-LNP vaccine formulated with a ms6 CO2 RNA construct with a TM modification (1 μg); or lyophilized VZV RNA-LNP vaccine formulated with gE_WT CO2 (gE_WT CO2 Lyo) (1 μg). FIG. 27C shows B cell responses assessed in splenocytes by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry.

DETAILED DESCRIPTION The present disclosure provides RNA molecules (e.g., RNA polynucleotides) that include at least one open reading frame (ORF) encoding a Varicella Zoster Virus (VZV) antigen. In some embodiments, the VZV antigen is a VZV polypeptide. In some embodiments, the VZV polypeptide is a VZV gE polypeptide. In some embodiments, the VZV polypeptide includes an amino acid sequence of Table 1. In some embodiments, the RNA molecule includes an ORF transcribed from at least one DNA nucleic acid sequence of Table 2. In some embodiments, the RNA molecule includes an ORF that includes an RNA nucleic acid sequence of Table 3. In some embodiments, the RNA molecule includes at least one of a 5' cap, a 5' UTR, a 3' UTR, and a poly A tail. The present disclosure provides RNA molecules (e.g., modified RNA; modRNA) that include modified nucleotides. The present disclosure provides an immunogenic composition comprising any one of the RNA molecules encoding a VZV polypeptide described herein complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forming a lipid nanoparticle (RNA-LNP). The present disclosure further provides an immunogenic composition comprising any one of the RNA molecules comprising at least one RNA nucleic acid described herein complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forming a RNA-LNP. The present disclosure further provides a method of preventing, treating, or ameliorating an infection, disease, or condition (e.g., herpes zoster or shingles) in a subject through administering to the subject an effective amount of an RNA molecule, RNA-LNP, or immunogenic composition described herein. The present disclosure further provides the use of the RNA molecule, RNA-LNP, and/or immunogenic composition described herein as a vaccine.

I. Example Definitions Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the art of cell and molecular biology to indicate a deviation of ±10% of the value with which it is placed.

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

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

The word "and/or" means "and" or "or." Illustratively, A, B, and/or C includes A alone, B alone, C alone, A in combination with B, A in combination with C, B in combination with C, or A in combination with B and C. In other words, "and/or" functions as an inclusive or.

The phrase "essentially all" is defined as "at least 95%"; if essentially all members of a group have a particular property, then at least 95% of the members of the group have that property. In some embodiments, essentially all means that equal to, at least, or between any two of 95, 96, 97, 98, 99, or 100% of the members of the group have that property.

The compositions and methods of their use may "comprise", "consist essentially of", or "consist" of any of the components or steps disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, the words "comprising" (as well as any form of comprising, e.g., "comprise" and "comprises"), "having" (as well as any form of having, e.g., "have" and "has"), "including" (as well as any form of including, e.g., "including" and "comprises"), "having" (as well as any form of having, e.g., "have" and "has"), "including" (as well as any form of including, e.g., "including" and "comprises"), "having" (as well as any form of including, e.g., "have" and "has"), "including" (as well as any form of including, e.g., "have" and "has"), "having ... It is understood that "includes" and "comprise" or "containing" (as well as any forms of containing, such as "contains" and "contain") are inclusive or open-ended, implying the inclusion of a recited 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. It is contemplated that aspects described herein in the context of the term "comprise" may also be implemented in the context of the term "consisting of" or "consisting essentially of." Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the scope of the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The word "consisting of" (as well as any forms of "consisting of", such as "consist of" and "consists of") are meant to include and be limited to everything that precedes the word "consisting of". Thus, the word "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present.

References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. As such, the appearances of such phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Moreover, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The terms "inhibit," "reduce," or "reduce" or any variation of these terms include any measurable reduction (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% reduction) or complete inhibition to achieve the desired result. The terms "enhance," "promote," or "increase," or any variation 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 a desired result or production of a protein or molecule.

As used herein, the terms "reference," "standard," or "control" describe a value to which a comparison is made. For example, an agent, subject, population, sample, or value of interest is compared to a reference, standard, or control agent, subject, population, sample, or value of interest. The reference, standard, or control may be tested and/or determined substantially simultaneously and/or in conjunction with the test or determination of interest for the agent, subject, population, sample, or value of interest, and/or may be determined or characterized under conditions or circumstances comparable to the agent, subject, population, sample, or value of interest being evaluated.

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

"Nucleic acid" as used herein refers to a molecule that contains a nucleic acid component 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 or consists of nucleotide monomers covalently linked to each other by sugar/phosphate backbone phosphodiester bonds. Nucleic acids may also include modified nucleic acid molecules, such as DNA or RNA molecules that are base-modified, sugar-modified or backbone-modified. Nucleic acids may be present in a variety of forms, such as isolated segments and integrated sequences or recombinant vectors of recombinant polynucleotides that encode a polypeptide, such as one or both chains of an antigen or antibody, or a fragment, derivative, mutein, or variant thereof, polynucleotides sufficient for use as hybridization probes, PCR primers or sequencing primers for identification, analysis, mutation, or amplification of a polynucleotide encoding a polypeptide, polynucleotides as described herein above, mRNA, saRNA, and antisense nucleic acids for inhibiting expression of complementary sequences. The nucleic acid may encode an epitope to which an antibody may bind.

The term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding entity. In some embodiments, an epitope is composed of 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 near each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearized).

Nucleic acids may be single-stranded or double-stranded and may include RNA and/or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids). In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow purification, transport, secretion, post-translational modification, or therapeutic benefit, such as targeting or efficacy, of the polypeptide. Tags or other heterologous polypeptides may be added to the modified polypeptide coding sequence, with "heterologous" referring to a polypeptide that is not the same as the modified polypeptide.

The term "polynucleotide" refers to a nucleic acid molecule that may be recombinant or that has been isolated from total genomic nucleic acid. Recombinant vectors, including oligonucleotides (nucleic acids of 100 residues or fewer in length), such as plasmids, cosmids, phages, and viruses, are included in the term "polynucleotide." Polynucleotides, in certain embodiments, include regulatory sequences that are substantially isolated from their naturally occurring gene or protein coding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

In certain embodiments, polynucleotide variants having substantial identity to the sequences disclosed herein; equal to or at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher percent sequence identity compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). %, 95%, 96%, 97%, 98%, or 99% or higher percent sequence identity, or up to 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher percent sequence identity, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher percent sequence identity. In certain embodiments, the isolated polynucleotide comprises a nucleotide sequence encoding a polypeptide having at least 90% identity to the amino acid sequence described herein over the entire length of the sequence; or a nucleotide sequence complementary to the isolated polynucleotide. In some embodiments, the isolated polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 95% identity over the entire length of the sequence to an amino acid sequence described herein; or a nucleotide sequence that is complementary to the isolated polynucleotide.

Nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, and other coding segments, so that their overall length may vary considerably. Nucleic acids may be of any length. They may be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, or may be at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 1000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length. 0, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, or up to 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length. or any one of 5, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length. , 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, and/or may include one or more additional sequences, e.g., regulatory sequences, and/or may be part of a larger nucleic acid, e.g., a vector. Thus, it is contemplated that a nucleic acid fragment of almost any length may be used, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.

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

As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product from the nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, expression of 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 a polypeptide or protein.

In general, the term "engineered" refers to an aspect that has been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when it is manipulated by the hand of man such that two or more sequences that are not linked in that order in nature are directly linked to one another in the engineered polynucleotide, and/or when certain residues in the polynucleotide are caused through the action of the hand of man to be linked to entities or moieties that do not occur in nature and/or are not linked in nature.

The term "DNA" as used herein means a nucleic acid molecule that includes nucleotides, e.g., deoxy-adenosine-monophosphate, deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate, and deoxy-cytidine-monophosphate monomers, that are composed of a sugar moiety (deoxyribose), a base moiety, and a phosphate moiety, and that polymerize with a characteristic backbone structure. The backbone structure is typically formed by a phosphodiester bond between the sugar moiety, e.g., deoxyribose, of a nucleotide of a first monomer and the phosphate moiety of a second adjacent monomer. The specific order of the monomers, e.g., the order of the bases linked to the sugar/phosphate backbone, is called the DNA sequence. DNA may be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand typically hybridize with the nucleotides of the second strand, e.g., by A/T and G/C base pairing. DNA may contain all, or mostly, deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. Without any limitation, DNA may include double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

The term "RNA" as used herein means a nucleic acid molecule comprising nucleotides, e.g., adenosine-monophosphate, uridine-monophosphate, guanosine-monophosphate and cytidine-monophosphate monomers, connected to one another along a so-called backbone. The backbone is formed by phosphodiester bonds between the sugar, e.g., ribose, of a first monomer and the phosphate moiety of a second adjacent monomer. The RNA may be obtained, for example, by transcription of a DNA sequence inside a cell. In eukaryotic cells, transcription typically takes place inside the nucleus or mitochondria. In vivo, transcription of DNA may result in an immature RNA that is processed into messenger RNA (mRNA). Processing of the immature RNA, e.g., in eukaryotes, includes various post-transcriptional modifications, e.g., splicing, 5'-capping, polyadenylation, export from the nucleus or mitochondria. The mature messenger RNA is processed to provide a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. A mature mRNA may include a 5' cap, a 5' UTR, an open reading frame, a 3' UTR and a poly-A tail sequence. The RNA may contain all or most of ribonucleotide residues. As used herein, the term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. In one embodiment, the RNA may be a messenger RNA (mRNA) for an RNA transcript that encodes a peptide or protein. As known to those skilled in the art, an mRNA generally contains a 5' untranslated region (5' UTR), a polypeptide coding region, and a 3' untranslated region (3' UTR). Without any limitation, the RNA may include double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA (modRNA).

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

"Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution, and/or alteration of one or more nucleotides compared to naturally occurring RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the 5' and/or 3' ends of the RNA. In one embodiment, such modRNA contains at least one modified nucleotide, e.g., an alteration to the base of a nucleotide. For example, a modified nucleotide may replace one or more uridine and/or cytidine nucleotides. For example, these replacements may occur for every instance of uridine and/or cytidine in the RNA sequence, or may occur only for select uridine and/or cytidine nucleotides. Such alterations to standard nucleotides in the RNA may include non-standard nucleotides, e.g., chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide may be replaced with N1-methylpseudouridine in the RNA sequence. Other such altered nucleotides are known to those of skill 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 may be a replicon RNA (replicon), particularly a self-replicating RNA, or a self-amplifying RNA (saRNA).

As contemplated herein, without any limitation, RNA may be used as a therapeutic modality to treat and/or prevent a number of conditions in a mammal, including a human. The methods described herein include administration of the RNA described herein to a mammal, e.g., a human. For example, in one embodiment, the method of using such RNA includes an antigen-encoding RNA vaccine to induce robust neutralizing antibodies and concomitant/concomitant T cell responses to achieve protective immunization. In some embodiments, a minimal vaccine dose is administered to induce robust neutralizing antibodies and concomitant/concomitant T cell responses to achieve protective immunization. In one embodiment, the administered RNA is an in vitro transcribed RNA. For example, such an RNA may be used to encode at least one antigen intended to generate an immune response in said mammal. The pathogenic antigen is a peptide or protein antigen derived from a pathogen associated with an infectious disease. In a specific embodiment, the pathogenic antigen is a peptide or protein antigen derived from VZV. Conditions and/or diseases that may be treated using the RNA disclosed herein include, but are not limited to, those caused and/or affected by viral infection. Such viruses include, but are not limited to, VZV.

"Prevent" or "prevention," as used herein when used in the context of the appearance of a disease, disorder, and/or condition, refers to reducing the risk of developing a disease, disorder, and/or condition and/or delaying the onset of one or more characteristics or symptoms of a disease, disorder, or condition. Prevention may be considered complete when the onset of a disease, disorder, or condition is delayed for a predefined period of time.

As will be understood from the context, "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 up to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 to 100%. In some embodiments, the risk is expressed as a risk relative to a 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 the disease, disorder, condition, and/or event. In some embodiments, the reference sample or group of reference samples is from an individual comparable to the particular individual. In some embodiments, the risk may reflect one or more genetic attributes, e.g., one or more genetic attributes that may predispose an individual to developing (or not developing) a particular disease, disorder, and/or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. Susceptibility: 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 members 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 develops the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition does not develop the disease, disorder, and/or condition.

The terms "protein," "polypeptide," or "peptide" are used synonymously herein and refer to a polymer of amino acid monomers, e.g., a molecule comprising at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments, and other equivalents, variants, and analogs of the above. Polypeptides may be single molecules or multi-molecular complexes, e.g., dimers, trimers, or tetramers. Proteins include one or more peptides or polypeptides and may be folded into a three-dimensional form that may be required for the protein to exert its biological function.

As used herein, the term "wild type" or "WT" or "native" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is used, while in other embodiments of the disclosure, a modified protein or polypeptide is used to generate an immune response. The above terms may be used interchangeably.

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

The term "fragment" in relation to an amino acid sequence (peptide or protein) relates to a portion of the amino acid sequence, e.g. a sequence representing an amino acid sequence truncated at the N-terminus and/or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon serving to initiate translation. A fragment of an amino acid sequence comprises, 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 this disclosure, a fragment of a polypeptide, DNA, nucleic acid, or RNA nucleic acid sequence is at least 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% identical to the polypeptide, DNA, nucleic acid, or RNA nucleic acid sequence from which it is derived, and up to 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%, or exactly 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%, or between any two of 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

In one embodiment, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has 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 that has 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 that has 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 that has 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 that has 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 that has at least 97% sequence identity to the polypeptide, DNA, nucleic acid, or RNA nucleic acid sequence from which it is derived. In one embodiment, a fragment of a polypeptide, DNA, nucleic acid, or RNA nucleic acid sequence refers to a sequence that has at least 99% sequence identity to the polypeptide, DNA, nucleic acid, or RNA nucleic acid sequence from which it is derived.

As used herein in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule, but that differs structurally from the reference molecule, e.g., in the presence or absence or level of one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant is, by definition, a distinct molecule that shares one or more such characteristic structural elements but differs 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 as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently constituent of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, the variant polypeptide or nucleic acid is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, up to 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, exactly 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%, or between any two of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, the variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to the reference amino acid or nucleotide sequence except for a small number of sequence changes 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. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In many cases, the variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of functional residue substitutions, insertions, or deletions compared to the reference. In some embodiments, 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 residue substitutions compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions.

In some embodiments, the reference polypeptide or nucleic acid is a "wild-type" or "WT" or "native" sequence found in nature, including allelic variations. A wild-type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant, and/or an amino acid substitution variant. A "variant" of a nucleotide sequence includes a nucleotide insertion variant, a nucleotide addition variant, a nucleotide deletion variant, and/or a nucleotide substitution variant. 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 in nature. The term "variant" specifically includes fragments of an amino acid or nucleic acid sequence.

Changes may be introduced into a nucleic acid by mutation, which may lead to a change in the amino acid sequence of a polypeptide (e.g., an antigen or an antibody or antibody derivative) that the nucleic acid encodes. Mutations may be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. In some embodiments, however made, the mutant polypeptide may be expressed and screened for desired properties.

Mutations may be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide it encodes. For example, nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues may be made. Alternatively, one or more mutations may be introduced into a nucleic acid that selectively alter the biological activity of the polypeptide it encodes. For example, a mutation may quantitatively or qualitatively alter a biological activity. Examples of quantitative changes include increasing, reducing or eliminating an activity. Examples of qualitative changes include altering the antigen specificity of an antibody.

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

The terms "% identical", "% identity" or similar terms are intended to specifically refer to the percentage of nucleotides or amino acids that are identical in optimal alignment between the sequences being compared. Said percentage is purely statistical, and the differences between the two sequences may, but need not, be randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "window of comparison" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm by Smith and Waterman, 1981, Ads App. Math. 2, 482, Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the search for similarity algorithm according to Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs using said algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). In some embodiments, the percent 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).

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

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

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

A fragment or variant of an amino acid sequence (peptide or protein) may be a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties identical or similar to one or more functional properties of the amino acid sequence from which it is derived, e.g., functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. The term "functional fragment" or "functional variant", as used herein, refers specifically to a variant molecule or sequence that comprises an amino acid sequence that is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, and that still has the ability to perform one or more of the functions of the parent molecule or sequence, e.g., the induction of an immune response. In one embodiment, the alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or significantly alter the characteristics of the molecule or sequence.

An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the first amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein may be altered such that their sequence varies from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

In this disclosure, a vector refers to a nucleic acid molecule, e.g., an artificial nucleic acid molecule. A vector may be used to incorporate a nucleic acid sequence, e.g., a nucleic acid sequence including an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. A vector may be an RNA vector or a DNA vector. In some embodiments, a vector is a DNA molecule. In some embodiments, a vector is a plasmid vector. In some embodiments, a vector is a viral vector. Typically, an expression vector contains a desired coding sequence and appropriate other sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are commonly used to manipulate and amplify a particular desired fragment (typically a DNA fragment) and may lack functional sequences required for expression of the desired fragment.

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

As used herein, the term "vaccination" refers to the administration of an immunogenic composition intended to generate an immune response, for example, against a disease-associated (e.g., disease-causing) agent (e.g., a virus). In some embodiments, vaccination may be administered before, during, and/or after exposure to a disease-associated agent, and in certain embodiments, before, during, and/or immediately after exposure to the agent. In some embodiments, vaccination involves multiple administrations of a vaccine composition, appropriately spaced in time. In some embodiments, vaccination generates an immune response against an infectious agent. In some embodiments, vaccination generates an immune response against a tumor; in some such embodiments, vaccination is "individualized" in that it is directed in part or in whole to epitopes (which may be or include, for example, one or more neo-epitopes) determined to be present in a particular individual's tumor.

Immune response refers to humoral, cellular, or both humoral and cellular responses in an organism. Immune response may be measured by assays 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 modulation in terms of activity or expression of one or more cytokines.

As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any dose of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administration of one or more agents or modalities to a subject who is receiving the other agent or modality in combination. For clarity, combination therapy does not require that the individual agents be administered together (or even necessarily simultaneously) in a single composition, although in some embodiments, the two or more agents, or active portions thereof, may be administered together in a combination composition or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

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

II. Varicella-zoster virus (VZV)
The present disclosure provides an RNA molecule (e.g., an RNA polynucleotide) that includes at least one open reading frame encoding a Varicella-Zoster Virus (VZV) polypeptide. The present disclosure further provides an immunogenic composition that includes at least one RNA molecule encoding a VZV polypeptide that is complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forms a lipid nanoparticle (LNP).

Varicella zoster virus (VZV), also known as human herpesvirus 3 (HHV-3), is a human pathogen that causes chickenpox or chicken pox in children and later re-emerges as herpes zoster or shingles. VZV has an inner capsid that encloses a linear double-stranded DNA genome. Surrounding the capsid is a tegument layer with glycoproteins, and the outermost layer is a lipid-rich envelope with glycoproteins. The glycoproteins have a variety of functions, from DNA replication or capsid assembly to interacting with cell surface molecules and assisting in fusion into the plasma membrane. For example, glycoprotein E is an integral membrane protein that is thought to be important for T-cell infection and cell-to-cell spread of the virus. VZV displays tropism for neurons and T cells.

Upon primary infection with VZV (e.g., chickenpox or "chicken pox"), VZV establishes latency in sensory ganglia. VZV-specific T cells are required to clear the primary infection and prevent reactivation. The mechanism of reactivation is unknown, but VZV cell-mediated immunity is thought to play a role. Deficiencies in cell-mediated immunity (e.g., advanced age, immunocompromised states) are risk factors for reactivation. Reactivation allows VZV replication and transport to the skin, where it may manifest as herpes zoster (HZ).

Herpes zoster most commonly appears as a painful unilateral vesicular rash typically confined to one dermatome or several contiguous dermatomes. Within a few days of the onset of the rash, grouped vesicles, bullae, or pustules may develop; these lesions contain VZV and are thought to be infectious. The characteristic pain of herpes zoster includes a burning or numbing sensation, pruritus, or allodynia. Many people develop prodromal pain 2-3 days before the rash appears. In immunocompetent people, the lesions crust over within 7-10 days and are no longer infectious once crusted.

The most common complication of herpes zoster is postherpetic neuralgia (PHN), which develops in up to 15% of people with herpes zoster. PHN is significant pain in the area affected by herpes zoster after the rash has crusted. Older age and prodrome are thought to be risk factors for PHN. Other complications of herpes zoster include ophthalmic complications (herpes zoster ophthalmic or keratitis, acute retinal necrosis), neurological complications (herpes zoster oticus, meningitis, encephalitis, myelitis, peripheral motor neuropathy, Guillain-Barré syndrome, and stroke), and secondary bacterial skin and soft tissue infections.

The VZV genome encodes at least 71 unique proteins (ORF0-ORF68) with three additional open reading frames (ORF69-ORF71) that overlap earlier open reading frames (ORF64-62, respectively). The encoded proteins form the structure of the virus particle, including nine glycoproteins: ORF5 (gK), ORF9A (gN), ORF14 (gC), ORF31 (gB), ORF37 (gH), ORF50 (gM), ORF60 (gL), ORF67 (gI), and ORF68 (gE). The encoded glycoproteins gE, gI, gB, gH, gK, gL, gC, gN, and gM function in different steps of the viral replication cycle. Besides infected cells, the most abundant glycoprotein found in mature virions is glycoprotein E (gE, ORF 68), which is the major component of the virion envelope and is essential for viral replication. Glycoprotein I (gI, ORG 67) forms a complex with gE in infected cells, which promotes endocytosis of both glycoproteins and targets them to the trans-Golgi network (TGN) where the final viral envelope is acquired. VZV gE is a 623 amino acid type I membrane protein encoded by open reading frame 68 (ORF68) and is the most abundant viral glycoprotein expressed on the surface of VZV-infected cells. Glycoprotein I (gI) is required for VZV envelope formation in the TGN and for efficient membrane fusion during VZV replication. VZV gE and gI are found complexed together on the surface of infected host cells. Glycoprotein B (ORF 31), the second most abundant glycoprotein and believed to play a role in viral entry, binds neutralizing antibodies. Glycoprotein H is believed to have a fusion function that facilitates cell-to-cell spread of the virus. Antibodies against gE, gB, and gH are abundant after natural infection and vaccination and have been shown to neutralize viral activity in vitro. As used herein, the term "varicella zoster virus" or "VZV" is not limited to any particular strain or variant.

In some embodiments, the RNA molecule comprises an open reading frame encoding a VZV antigen. In some embodiments, the VZV antigen is a VZV polypeptide. In some embodiments, the VZV polypeptide is a VZV glycoprotein (e.g., gK, gN, gC, gB, gH, gM, gL, gI, and gE) or a fragment or variant thereof. In some embodiments, the RNA molecule encodes a VZV gK polypeptide, the RNA molecule encodes a VZV gN polypeptide, the RNA molecule encodes a VZV gC polypeptide, the RNA molecule encodes a VZV gB polypeptide, the RNA molecule encodes a VZV gH polypeptide, the RNA molecule encodes a VZV gM polypeptide, the RNA molecule encodes a VZV gL polypeptide, the RNA molecule encodes a VZV gI polypeptide, and/or the RNA molecule encodes a VZV gE polypeptide. In one embodiment, the RNA molecule encodes a VZV gE polypeptide. In some embodiments, the VZV polypeptide comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) VZV polypeptides.

In some embodiments, the VZV polypeptide is a full-length VZV polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV polypeptide.

In some embodiments, the VZV polypeptide is a full-length gK polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gK polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gK polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gK polypeptide.

In some embodiments, the VZV polypeptide is a full-length gN polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gN polypeptide. In some embodiments, the VZV polypeptide is a variant of the VZV gN polypeptide. In some embodiments, the VZV polypeptide is a fragment of the VZV gN polypeptide.

In some embodiments, the VZV polypeptide is a full-length gC polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gC polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gC polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gC polypeptide.

In some embodiments, the VZV polypeptide is a full-length gB polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gB polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gB polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gB polypeptide.

In some embodiments, the VZV polypeptide is a full-length gH polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gH polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gH polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gH polypeptide.

In some embodiments, the VZV polypeptide is a full-length gM polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gM polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gM polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gM polypeptide.

In some embodiments, the VZV polypeptide is a full-length gL polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gL polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gL polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gL polypeptide.

In some embodiments, the VZV polypeptide is a full-length gI polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gI polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gI polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gI polypeptide.

In some embodiments, the VZV polypeptide is a full-length gE polypeptide. In some embodiments, the VZV polypeptide is a truncated VZV gE polypeptide. In some embodiments, the VZV polypeptide is a variant of a VZV gE polypeptide. In some embodiments, the VZV polypeptide is a fragment of a VZV gE polypeptide.

In some embodiments, the VZV polypeptide comprises at least one mutation. In some embodiments, the VZV polypeptide is a VZV gK polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gN polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gC polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gB polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gH polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gM polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gL polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gI polypeptide comprising at least one mutation. In some embodiments, the VZV polypeptide is a VZV gE polypeptide comprising at least one mutation.

In some embodiments, the RNA molecule comprises a VZV comprising an amino acid sequence according to any one of GENBANK® Accession Nos.: AAG32558.1, ABE03086.1, AAK01047.1, Q9J3M8.1, AEW88548.1, AGY33616.1, AEW89124.1, AIT53150.1, CAA25033.1, NP_040190.1, AKG56356.1, AEW89412.1, ABF21714.1, ABF21714.1, AAT07749.1, AEW88764.1, AAG48520.1, and/or AEW88980.1, each of which sequences is incorporated herein by reference. In some embodiments, the RNA molecule encodes a VZV gE polypeptide, or a fragment or variant thereof, comprising an amino acid sequence according to GENBANK® Accession No. AH009994.2 (ORF68), the sequence of which is incorporated herein by reference.

In some embodiments, the RNA molecule encodes a VZV polypeptide of Table 1 (see Example 7). In some embodiments, the RNA molecule encodes a VZV gE polypeptide comprising an amino acid sequence of any of SEQ ID NOs: 1-11, or a fragment or variant thereof. In some embodiments, the VZV gE polypeptide has a sequence similar to any of the amino acid sequences of Table 1, e.g., any of SEQ ID NOs: 1-11, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1101%, 1102%, 1103%, 1104%, 1105%, 1106%, 1107%, 1108%, 1109%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 3%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 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%, exactly 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%, or 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%, %, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity between any two of the sequences. In some embodiments, the VZV gE polypeptide consists of any of the amino acid sequences in Table 1, e.g., any of SEQ ID NOs: 1-11.

In some embodiments, the RNA molecule sequence is transcribed from a DNA nucleic acid sequence (DNA polynucleotide) of Table 2 (see Example 7). In some embodiments, the RNA molecule comprises an ORF transcribed from any of the nucleic acid sequences of SEQ ID NOs: 12-145, or a fragment or variant thereof. In some embodiments, the RNA molecule comprises an ORF transcribed from any of the nucleic acid sequences of Table 2, e.g., any of SEQ ID NOs: 12-145, that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145, 145, 145, 145, 146%, 147%, 148%, 149 93%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 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%, exactly 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%, or 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%. The ORF may have between any two of the following identities: 3%, 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 of the nucleic acid sequences in Table 2, e.g., any of SEQ ID NOs: 12-145.

In some embodiments, the RNA molecule comprises an ORF comprising an RNA nucleic acid sequence (RNA polynucleotide) of Table 3 (see Example 7). In some embodiments, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any of SEQ ID NOs: 146-279, or a fragment or variant thereof. In some embodiments, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any of SEQ ID NOs: 146-279, or a fragment or variant thereof. In some embodiments, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any of SEQ ID NOs: 146-279, or a fragment or variant thereof. 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, up to 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%, exactly 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%, or 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%, The ORF may have between any two of the following 2%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. In some embodiments, the RNA molecule comprises an ORF that includes a nucleic acid sequence consisting of any of the RNA nucleic acid sequences in Table 3, e.g., any of SEQ ID NOs: 146-279.

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 by N1-methylpseudouridine. In some embodiments, the RNA molecule comprises a sequence in which all uridines are replaced by N1-methylpseudouridine (designated as "Ψ"). In some embodiments, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any of SEQ ID NOs: 146-279 in which all uridines are replaced by N1-methylpseudouridine (designated as "Ψ").

In some embodiments, the RNA molecule is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the VZV polypeptide sequences of SEQ ID NOs: 1-11 (Table 1) or other VZV polypeptides described herein, and up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the VZV polypeptide sequences of SEQ ID NOs: 1-11 (Table 1) or other VZV polypeptides described herein. or 99%, or exactly 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or between any two of 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the RNA molecule comprises an open reading frame encoding a VZV polypeptide amino acid sequence consisting of any of the VZV polypeptide sequences of SEQ ID NOs: 1-11 (Table 1) or other VZV polypeptides described herein.

In some embodiments, the RNA molecule is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of SEQ ID NOs: 12-145 (Table 2) or other nucleic acids described herein, and up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of SEQ ID NOs: 12-145 (Table 2) or other nucleic acids described herein. comprises an open reading frame transcribed from a DNA nucleic acid sequence that may be 99%, exactly 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or between any two of 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence consisting of any of the nucleic acid sequences of SEQ ID NOs: 12-145 (Table 2) or other nucleic acids described herein.

In some embodiments, the RNA molecule is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of SEQ ID NOs: 146-279 (Table 3) or other nucleic acids described herein, and up to 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the nucleic acid sequences of SEQ ID NOs: 146-279 (Table 3) or other nucleic acids described herein. or 99%, or exactly 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or between any two of 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% percent identical. In some embodiments, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence consisting of a nucleic acid sequence of SEQ ID NOs: 146-279 (Table 3) or any of the other nucleic acids described herein. In some embodiments, the RNA molecule comprises an ORF comprising any of the nucleic acid sequences of SEQ ID NOs: 146-279 (Table 3) in which all uridines are replaced by N1-methylpseudouridine (designated as "Ψ").

III. RNA molecules In some embodiments, the RNA molecules described herein are coding RNA molecules. Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some embodiments, coding RNA molecules include at least one open reading frame (ORF) that encodes at least one peptide or polypeptide. An open reading frame includes a sequence of codons that can be translated into a peptide or protein. Coding RNA molecules may include one (monocistronic), two (bicistronic) or more (multicistronic) ORFs, which may be a sequence of codons that can be translated into a polypeptide or protein of interest.

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

An RNA molecule may encode one or more polypeptides of interest, e.g., one or more antigens, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more polypeptides. Alternatively, or in addition, an RNA molecule may also encode more than one polypeptide of interest, e.g., an antigen, e.g., a bicistronic or tricistronic RNA molecule encoding different or identical antigens.

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

The term "codon-optimized" is understood by those skilled in the art to refer to altering codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of the present disclosure, in some embodiments, the coding region is codon-optimized for optimal expression in a subject treated with an RNA polynucleotide described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNA molecules in a cell. Thus, the sequence of the RNA may be modified such that codons available for frequently occurring tRNA molecules are inserted in place of "rare codons."

In some embodiments, the G/C content of the coding region of the RNA (e.g., the sequence of the gene of interest; the 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, and in some embodiments, the amino acid sequence encoded by the RNA is not modified 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 the efficient translation of its mRNA. Sequences with an increased G (guanosine)/C (cytidine) content are more stable than sequences with an increased A (adenosine)/U (uridine) content. With regard to the fact that several codons code for one and the same amino acid (the so-called degeneracy of the genetic code), the most favorable codons for stability may be determined (the so-called alternative codon usage). 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 nucleosides may be modified by replacing these codons with other codons that encode the same amino acids but that do not contain A and/or U or that contain a lower content of A and/or U nucleosides. Thus, in some embodiments, the G/C content of the coding region of the RNA described herein is increased by at least 10%, 20%, 30%, 40%, 50%, 55%, up to 10%, 20%, 30%, 40%, 50%, 55%, exactly 10%, 20%, 30%, 40%, 50%, 55%, or between any two of 10%, 20%, 30%, 40%, 50%, 55%, or even higher percentages compared to the G/C content of the coding region of the wild-type RNA. In some embodiments, the coding regions of the VZV RNA described herein comprise 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 regions of the VZV RNA described herein comprise 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 regions of the VZV RNA described herein comprise 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 regions of the VZV RNA described herein comprise a G/C content of about 58%, about 66%, or about 62%.

In some embodiments, the RNA molecule is 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).

In some embodiments, the RNA molecule is at least 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, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 420 0, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 660 0,6800,7000,7200,7400,7600,7800,8000,8200,8400,8600,8800,9000,9200,9400,9600,9800,10000,10000,12000,14000,16000,18000,200 00, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 400 00, 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 100,000 pieces, up to 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, 88 0, 900, 920, 940, 960, 980, 1000, 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, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24 000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44 000, 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, exactly 2 0, 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, 64 0, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 94 0,960,980,1000,1000,1200,1400,1600,1800,2000,2200,2400,2600,2800,3000,3200,3400,3600,3800,4000,4200,4400,4600,4800,5000,5 200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7 600,7800,8000,8200,8400,8600,8800,9000,9200,9400,9600,9800,10000,10000,12000,14000,16000,18000,20000,22000,24000,26000,28 000, 30000, 32000, 34000, 36000, 38000, 40000, 42000, 44000, 46000, 48 000, 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, or about 20, 40, 60, 80, 100,120,140,160,180,200,220,240,260,280,300,320,340,360,380,400,420,440,460,480,500,520,540,560,580,600,620,640,660,680,7 00, 720, 740, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1 000,1000,1200,1400,1600,1800,2000,2200,2400,2600,2800,3000,3200,3400,3600,3800,4000,4200,4400,4600,4800,5000,5200,5400,56 00, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 80 00,8200,8400,8600,8800,9000,9200,9400,9600,9800,10000,10000,12000,14000,16000,18000,20000,22000,24000,26000,28000,30000,3 2000, 34000, 36000, 38000, 40000, 42000, 44000, 46000, 48000, 50000, 5 It has a number of nucleotides between any two of 2000, 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.

In some embodiments, the RNA molecule comprises at least 100 nucleotides. For example, in some embodiments, the RNA has a length of 100-15,000 nucleotides; 7,000-16,000 nucleotides; 8,000-15,000 nucleotides; 9,000-12,500 nucleotides; 11,000-15,000 nucleotides; 13,000-16,000 nucleotides; 7,000-25,000 nucleotides. In some embodiments, the RNA molecule comprises at least 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, 31 00,3150,3200,3250,3300,3350,3400,3450,3500,3550,3600,3650,3700,3750,3800,3850,3900,3950,4000,4050,4100,4150,4200,4250,430 0, 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, 5 850, 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, 77 00,7750,7800,7850,7900,7950,8000,8050,8100,8150,8200,8250,8300,8350,8400,8450,8500,8550,8600,8650,8700,8750,8800,8850,890 0, 8950, 9000, 9050, 9100, 9150, 9200, 9 250,9300,9350,9400,9450,9500,9550,9600,9650,9700,9750,9800,9850,9900,9950,10000,10050,10100,10150,10200,10250,10300,10350 , 10400, 10450, 10500, 10550, 10600, 1 0650,10700,10750,10800,10850,10900,10950,11000,11050,11100,11150,11200,11250,11300,11350,11400,11450,11500,11550,11600,11 650, 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, 13 200, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 142 00, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 pieces, up to 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 10 50,1100,1150,1200,1250,1300,1350,1400,1450,1500,1550,1600,1650,1700,1750,1800,1850,1900,1950,2000,2050,2100,2150,2200,225 0, 2300, 2350, 2400, 2450, 2500, 2550, 2 600,2650,2700,2750,2800,2850,2900,2950,3000,3050,3100,3150,3200,3250,3300,3350,3400,3450,3500,3550,3600,3650,3700,3750,38 00, 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, 565 0, 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, 7 200,7250,7300,7350,7400,7450,7500,7550,7600,7650,7700,7750,7800,7850,7900,7950,8000,8050,8100,8150,8200,8250,8300,8350,84 00, 8450, 8500, 8550, 8600, 8650, 8700, 8750,8800,8850,8900,8950,9000,9050,9100,9150,9200,9250,9300,9350,9400,9450,9500,9550,9600,9650,9700,9750,9800,9850,9900,9 950, 10000, 10050, 10100, 10150, 1020 0,10250,10300,10350,10400,10450,10500,10550,10600,10650,10700,10750,10800,10850,10900,10950,11000,11050,11100,11150,11200 , 11250, 11300, 11350, 11400, 11450, 11 500,11550,11600,11650,11700,11750,11800,11850,11900,11950,12000,12050,12100,12150,12200,12250,12300,12350,12400,12450,125 00, 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, 140 50, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 pieces, exactly 100, 150, 200, 250, 300, 350, 400, 450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,1250,1300,1350,1400,1450,1500,1550,1600,1650,1700,17 50, 1800, 1850, 1900, 1950, 2000, 2050, 2100,2150,2200,2250,2300,2350,2400,2450,2500,2550,2600,2650,2700,2750,2800,2850,2900,2950,3000,3050,3100,3150,3200,3250,3 300, 3350, 3400, 3450, 3500, 3550, 360 0, 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, 51 50,5200,5250,5300,5350,5400,5450,5500,5550,5600,5650,5700,5750,5800,5850,5900,5950,6000,6050,6100,6150,6200,6250,6300,635 0, 6400, 6450, 6500, 6550, 6600, 6650, 6700,6750,6800,6850,6900,6950,7000,7050,7100,7150,7200,7250,7300,7350,7400,7450,7500,7550,7600,7650,7700,7750,7800,7850,7 900, 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, 97 50, 9800, 9850, 9900, 9950, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10 800, 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, 12 350,12400,12450,12500,12550,12600,12650,12700,12750,12800,12850,12900,12950,13000,13050,13100,13150,13200,13250,13300,133 50, 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, 1 4650, 14700, 14750, 14800, 14850, 149 00, 14950, or 15000, or 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, 155
0,1600,1650,1700,1750,1800,1850,1900,1950,2000,2050,2100,2150,2200,2250,2300,2350,2400,2450,2500,2550,2600,2650,2700,2750 , 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 34 00,3450,3500,3550,3600,3650,3700,3750,3800,3850,3900,3950,4000,4050,4100,4150,4200,4250,4300,4350,4400,4450,4500,4550,460 0, 4650, 4700, 4750, 4800, 4850, 4900, 4950, 5000, 5050, 5100, 5150, 5200, 52 50,5300,5350,5400,5450,5500,5550,5600,5650,5700,5750,5800,5850,5900,5950,6000,6050,6100,6150,6200,6250,6300,6350,6400,645 0, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 71 00,7150,7200,7250,7300,7350,7400,7450,7500,7550,7600,7650,7700,7750,7800,7850,7900,7950,8000,8050,8100,8150,8200,8250,830 0, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 89 50,9000,9050,9100,9150,9200,9250,9300,9350,9400,9450,9500,9550,9600,9650,9700,9750,9800,9850,9900,9950,10000,10050,10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 1065 0,10700,10750,10800,10850,10900,10950,11000,11050,11100,11150,11200,11250,11300,11350,11400,11450,11500,11550,11600,11650 , 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 122 00,12250,12300,12350,12400,12450,12500,12550,12600,12650,12700,12750,12800,12850,12900,12950,13000,13050,13100,13150,1320 0, 13250, 13300, 13350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000 nucleotides.

In some aspects of the present disclosure, the RNA is or comprises a messenger RNA (mRNA) for an RNA transcript that encodes a polypeptide. In some aspects, the RNA disclosed herein comprises a 5' cap comprising a 5' cap as disclosed herein; a 5' untranslated region (5'UTR) comprising a cap-proximal sequence, a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3'UTR); and/or a polyadenylation (polyA) sequence.

In some embodiments, the RNA disclosed herein comprises the following components in a 5' to 3' orientation: a 5' cap comprising a 5' cap disclosed herein; a 5' untranslated region (5'UTR) comprising a cap-proximal sequence, a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3'UTR); and a polyA sequence.

A. Modified nucleobases In the present disclosure, RNA molecules may include modified nucleosides and modified nucleobases that can be incorporated into nucleotides.In some embodiments, RNA molecules may include one or more modified nucleotides.Naturally occurring nucleotide modifications are known in the art.

In some embodiments, the RNA molecule may include modified nucleotides. Non-limiting examples of modified nucleotides that may be included in the 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-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxy ... 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-taurinomethyl-uridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine 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 These include o-uridine, a-thio-uridine, 2'-O-methyl-uridine, 5,2'-O-dimethyl-uridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-carbamoylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, any other modified uridine known in the art, or combinations thereof.

In some aspects of the present disclosure, the modified nucleotide comprises one of N1-methylpseudouridine or pseudouridine.

In some embodiments, the RNA molecule comprises N1-methylpseudouridine modified nucleotides. In some embodiments, the RNA molecule comprises pseudouridine modified nucleotides.

In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine. In some embodiments, the RNA molecule comprises a sequence in which at least one uridine is replaced by N1-methylpseudouridine. In some embodiments, the RNA molecule comprises a sequence in which all uridines are replaced by N1-methylpseudouridine. N1-methylpseudouridine is designated in the sequence as "Ψ". The term "uracil" as used herein describes one of the nucleobases that may be present in an RNA nucleic acid. The term "uridine" as used herein describes one of the nucleosides that may be present in an RNA. "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine, in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

In some embodiments, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced by N1-methylpseudouridine or pseudouridine. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced by N1-methylpseudouridine or pseudouridine. %, 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%, most Large: 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%, exactly 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%, or 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12% , 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the uridines are replaced by N1-methylpseudouridine or pseudouridine. In some embodiments, the RNA molecule comprises a nucleic acid sequence in which all uridines are replaced by N1-methylpseudouridine or pseudouridine.

Modifications that may be present in an RNA molecule include, for example, m5C (5-methylcytidine), m5U (5-methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-O-methyluridine), m1A (1-methyladenosine); m2A (2-methyladenosine); Am (2-1-O-methyladenosine); ms2m6A (2-methylthio-N6-methyl adenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycinylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); Ar(p) (2'-O-ribosyladenosine); ms2t6A (2-methylthio-N6-threonylcarbamoyl adenosine); m6t6A (N6-methyl-N6-threonylcarbamoyl adenosine); hn6A (N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine); Ar(p) (2'-O-ribosyladenosine); I (inosine); mil (1-methylinosine); m'lm (1,2'-O-dimethylinosine); m3C (3-methylcytidine); Cm (2T-O-methylcytidine); s2C (2-thiocytidine); ac4C (N4-acetylcytidine); f5C (5-formylcytosine); m5Cm (5,2-O-dimethylcytidine); ac4Cm (N4 acetyl 2 TO-methylcytidine); k2C (lysine); m1G (1-methylguanosine); m2G (N2-methylguanosine); m7G (7-methylguanosine); Gm (2'-O-methylguanosine); m22G (N2,N2-dimethylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); Gr(p) (2'- O-ribosylguanosine (phosphate); yW (wybutosine); o2yW (peroxywybutosine); OHYW (hydroxywybutosine); OHYW* (hypomodified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (quenosine); oQ (epoxyquenosine); galQ (galactosyl-quenosine); manQ (mannosyl-quenosine). syn); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-O-dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2-thiouridine); s2Um (2-thio-2'-O-methyluridine); acp3U (3-(3-amino-3-carboxypropyl)uridine); ho5U (5-hydroxyuridine); mo5U (5-methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5-(carboxyhydroxymethyl)uridine); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mc m5U (5-methoxycarbonylmethyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyluridine); ncm5Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm5U (5-carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-L-O-methyluridine); m6Am (N6,T-O-dimethyladenosine); m62A (N6,N6-dimethyladenosine); Tm (2'-O-methylinosine); m4C (N4-methylcytidine); m4Cm (N4,2-O-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-O-dimethyladenosine); rn62Am (N6,N6,O-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m3Um (3,2T-O-dimethyluridine); m5D (5-methyldihydrouridine); f5Cm (5-formyl-2'-O-methylcytidine); m1Gm (1,2'-O-dimethylguanosine) m'Am (1,2-O-dimethyladenosine) irinomethyluridine); tm5s2U (S-taurinomethyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thiouridine, uracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(C1-C6)-alkylcytosine, 5 -methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7-deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza-8-substituted guanine, 8-hydroxycytosine, 8-hydroxy- Further includes cytoguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6-chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U.

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

The sequence of an RNA molecule may be modified, if desired, for example to increase the efficiency of expression or replication of the RNA, or to provide additional stability or resistance to degradation. For example, an RNA sequence may be modified with respect to its codon usage, for example to increase the translation efficiency and half-life of the RNA.

In some embodiments, the RNA molecules of the present disclosure include an open reading frame having at least one codon-modified sequence. A codon-modified sequence refers to a coding sequence that differs in at least one codon (a triplet of nucleotides that codes for one amino acid) compared to the corresponding wild-type coding sequence. The codon-modified sequence may exhibit improved resistance to degradation, improved stability, and/or improved translatability.

The sequence of the RNA molecule may be codon-optimized or deoptimized for expression in a desired host, e.g., a human cell.

In some embodiments, the RNA molecule may contain one or more structural and/or chemical modifications or alterations that confer useful properties to the polynucleotide, including, in some embodiments, lack of substantial induction of an innate immune response of a cell into which the polynucleotide 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 or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Since chemical bonds are necessarily broken and reformed to affect the structural modification, the structural modification is of a chemical nature and is therefore a chemical modification. However, the structural modification results in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG", where the dinucleotide "CC" is inserted, resulting in a structural modification to the polynucleotide.

In some embodiments, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. Naturally occurring nucleotide modifications are known in the art.

In some embodiments, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, with the exception of an optional 5' cap, which may include, e.g., 7-methylguanosine, as described further below. In some embodiments, the RNA may include a 5' cap that includes 7'-methylguanosine, and the first one, two, or three 5' ribonucleotides may be methylated at the 2' position of the ribose.

In some embodiments, the RNA molecules described herein are non-coding RNA molecules. Non-coding RNA (ncRNA) molecules include functional RNA molecules that are not translated into peptides or polypeptides. Non-coding RNA molecules may include highly abundant and functionally important RNA molecules. In some embodiments, the non-coding RNA is a functional mRNA molecule that is not translated into peptides or polypeptides. Non-coding RNA may include modified nucleotides as described herein. Preferably, the RNA molecule is an mRNA.

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

In some embodiments, the RNA molecules of the present disclosure are purified, e.g., by filtration, which may be performed, e.g., via ultrafiltration, diafiltration, or tangential flow ultrafiltration/diafiltration.

In some embodiments, the RNA molecules of the present disclosure are lyophilized to render them temperature stable.

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

In some embodiments, the 5' cap moiety is a natural 5' cap. A "natural 5' cap" is defined as a cap comprising a 7-methylguanosine attached to the 5' end of an mRNA molecule through a 5'-5' triphosphate linkage. In some embodiments, the guanosine nucleoside comprised in the 5' cap may be modified, for example, by methylation at one or more positions (e.g., position 7) on the base (guanine) and/or by methylation at one or more positions on the ribose. In some embodiments, the guanosine nucleoside comprised in the 5' cap comprises a 3'O methylation on the ribose (3'OMeG). In some embodiments, the guanosine nucleoside comprised in the 5' cap comprises a methylation at position 7 of the guanine (m7G). In some embodiments, the guanosine nucleoside comprised in the 5' cap comprises a methylation at position 7 of the guanine and a 3'O methylation on the ribose (m7(3'OMeG)). The 5' cap may be incorporated during RNA synthesis (e.g., transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some embodiments, transcriptional capping with a cap disclosed herein improves the capping efficiency of the RNA compared to transcriptional capping with a suitable reference comparator. In some embodiments, improved capping efficiency may increase the translation efficiency and/or translation rate of the RNA and/or increase expression of the encoded polypeptide. In some embodiments, capping is performed after purification of the RNA molecule, e.g., tangential flow filtration.

In some embodiments, the RNA described herein includes a 5' cap or a 5' cap analog, e.g., cap 0, cap 1, or cap 2. In some embodiments, the RNA provided 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 may be capped with a 5' cap analog. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0) and 7mG(5')ppp(5')N1mpNp (cap 1). In some embodiments, the RNA described herein includes cap 0. Cap 0 is an N7-methylguanosine connected to the 5' nucleotide through a 5'-5' triphosphate linkage, typically referred to as m7G cap or m7Gppp. In cells, the cap 0 structure is essential for efficient translation of capped mRNAs. Additional methylation at the 2'O position of the starting nucleotide produces what is referred to as Cap 1, or m7GpppNm, where Nm represents any nucleotide with a 2'O methylation. In some embodiments, the RNAs described herein include Cap 1, e.g., as described herein. In some embodiments, the RNAs described herein include Cap 2.

In some embodiments, the cap 0 structure comprises a guanosine nucleoside methylated at the 7 position of the guanine (m7G). In some embodiments, the cap 0 structure is connected to the RNA via a 5'-5'-triphosphate linkage, also referred to herein as m7Gppp or m7G(5')ppp(5'). The 5' cap may be methylated with the structure m7G(5')ppp(5')N (cap-0 structure) or a derivative thereof, where N is the terminal 5' nucleotide of a nucleic acid bearing a 5' cap, typically the 5' end of an mRNA. An exemplary enzymatic reaction for capping may include the use of vaccinia virus capping enzyme (VCE), which includes mRNA triphosphatase, guanylyl-transferase, and guanine-7-methyltransferase to catalyze the construction of the N7-monomethylated cap 0 structure. The cap 0 structure plays an important role in maintaining the stability and translational efficacy of RNA molecules.

The 5' cap of an RNA molecule may be further modified by a 2'-O-methyltransferase resulting in the generation of a cap 1 structure (m7Gppp[m2'-O]N), which may further increase translation efficiency. In some embodiments, the cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of the guanine (m7G) and a 2'O-methylated first nucleotide in the RNA (2'OmeN ₁ ). In some embodiments, the cap 1 structure is connected to the RNA via a 5'-5'-triphosphate linkage, also referred to herein as m7Gppp(2'OMeN ₁ ) 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, the m7G(5')ppp(5')(2'OmeN ₁ ) Cap 1 structure includes 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.

In some embodiments, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7 position of the guanine (m7G) and one or more additional modifications, e.g., a methylation on the ribose, and a 2'O-methylated first nucleotide in the RNA. In some embodiments, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7 position of the guanine, a 3'O-methylation on the ribose (m7(3'OMeG)), and a 2'O-methylated first nucleotide in the RNA (2'OMeN ₁ ). In some embodiments, the Cap 1 structure is connected to the RNA via a 5'-5'-triphosphate linkage, also referred to herein as m7(3'OMeG)ppp(2'OMeN ₁ ) 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 comprises 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, the second nucleotide in the Cap 1 structure may include one or more modifications, e.g., methylation. In some embodiments, a Cap 1 structure that includes a second nucleotide that includes a 2'O methylation is a Cap 2 structure.

In some embodiments, RNA molecules may be enzymatically capped at the 5' end using vaccinia guanylyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to yield a cap 0 structure. An inverted 7-methylguanosine cap is added via a 5'-5' triphosphate bridge. Alternatively, use of 2'O-methyltransferase with vaccinia guanylyltransferase yields a cap 1 structure in which, in addition to the cap 0 structure, the 2'OH group is methylated at the penultimate nucleotide. S-adenosyl-L-methionine (SAM) is a cofactor utilized as a methyl transfer reagent. Non-limiting examples of 5' cap structures are those that have, among others, enhanced binding of cap-binding polypeptides, increased half-life, reduced susceptibility to 5' endonucleases, and/or reduced 5' decapping compared to synthetic 5' cap structures (or wild-type, natural, or physiological 5' cap structures) known in the art.

For example, recombinant vaccinia virus capping enzyme and recombinant 2' O-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is referred to as a Cap 1 structure. This cap results in greater translational competence and cellular stability and reduced activation of cellular pro-inflammatory cytokines, for example, compared to other 5' cap analog structures known in the art.

In some embodiments, the 5' end cap comprises a cap analog, for example, the 5' end cap may comprise a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

In some embodiments, the capping region may comprise a single cap or a series of nucleotides that form a cap. In this embodiment, the capping region may be 1-10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In this embodiment, the capping region is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or a number between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, there is no cap. In some embodiments, the first and second functional regions may be in the range of 3-40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length, and may include one or more signal and/or restriction sequences in addition to start and/or stop codons. , at most 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, exactly 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, 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 in length, or between any two of 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 in length, and may include one or more signal and/or restriction sequences in addition to start and/or stop codons.

Further examples of 5' cap structures include glyceryl, inverted deoxy abasic residues (moieties), 4',5' methylene nucleotides, 1-(beta-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, non- These include, but are not limited to, cyclic 3,5 dihydroxypentyl nucleotides, 3'-3' inverted nucleotide moieties, 3'-3' inverted abasic moieties, 3'-2' inverted nucleotide moieties, 3'-2' inverted abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moieties.

In some embodiments, the RNA molecules of the present disclosure include at least one 5' cap structure. In some embodiments, the RNA molecules of the present disclosure do not include a 5' cap structure.

In one embodiment, the 5' capping structure comprises a modified 5' Cap 1 structure (m ⁷ G ⁺ m ^3' -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 in that it begins with a guanosine that is methylated at N7 and is linked to the first encoded nucleotide of the transcribed RNA (in this case, adenosine) by a 5'-5' triphosphate linkage. 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 to impart the Cap 1 structure.

C. Untranslated region (UTR)
A 5'UTR is a regulatory region located at the 5' end of a protein open reading frame that is transcribed into mRNA but not translated into an amino acid sequence, or the corresponding region in an RNA polynucleotide, e.g., an mRNA molecule. An open reading frame may be located 5' (upstream) of the open reading frame (5'UTR) and/or 3' (downstream) of the open reading frame (3'UTR).

In some embodiments, the UTRs are derived from mRNAs that are naturally abundant in the specific tissue (e.g., lymphoid tissue) where mRNA expression is targeted. In some embodiments, the UTRs increase protein synthesis. Without being bound by mechanism or theory, the UTRs may increase protein synthesis by increasing the time that the mRNA remains in the translation polysome (message stability) and/or the rate at which ribosomes begin translation on the message (message translation efficiency). Thus, the UTR sequence may prolong protein synthesis in a tissue-specific manner.

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 an mRNA that is naturally abundant in a tissue. The tissue may be, for example, liver, stem cells, or lymphoid tissue. The lymphoid tissue may include, for example, any one of lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, 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.

i. 5'UTR
In some embodiments, the RNA disclosed herein comprises a 5'UTR. The 5'UTR, if present, is located at the 5' end and initiates with a transcription start site upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5'cap (if present), e.g., directly adjacent to the 5'cap. The 5'UTR may contain various regulatory elements, e.g., a 5'cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in controlling translation initiation.

In some embodiments, a 5'UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some embodiments, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some embodiments, the cap-proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

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

One of skill in the art, upon reading this disclosure, 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) may be included in the RNA by reason of being included in a cap entity (e.g., such as a Cap1 structure); alternatively, in some embodiments, at least a portion of the residues in the cap-proximal sequence may be added enzymatically (e.g., by a polymerase, such as T7 polymerase). For example, in one particular exemplary embodiment in which a (m ₂ ^7,3'-O )Gppp(m ^{2 '-O} )ApG cap is 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).

In some embodiments, the cap-proximal sequence comprises _N1 and/or _N2 of the cap structure, where _N1 and _N2 are any nucleotide, e.g., A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U. In some embodiments, the cap-proximal sequence comprises _N1 and _N2 and _N3 , _N4 , and _N5 of the cap structure, where _N1 - _N5 correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 are any nucleotide, e.g., A, C, G, or U. In some embodiments, _N1N2 comprises 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, _N1N2 comprises AG and _N3N4N5 comprises 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.

In some embodiments, the cap _- proximal sequence comprises a sequence that includes _N1 and _N2 of the cap structure, and _A3A4X5 (SEQ ID NO:307; _X5 is A, G, C, or U), where _N1 and _N2 are each independently selected from A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _X5 is selected from A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. _In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the cap-proximal sequence comprises a sequence that includes _N1 and _N2 of the cap structure, and _C3A4X5 (SEQ ID NO _: 308; _X5 is A, G, C, or U), where _N1 and _N2 are each independently selected from A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _X5 is selected from A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. _In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the cap-proximal sequence comprises a sequence that includes the cap structure _{N1 and N2} , and _X3Y4X5 (SEQ ID NO:309; _{X3 or X5} are each independently selected from A, G, C, or U; and _Y4 is not C). In some embodiments, _N1 and _N2 are each independently selected from A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _X3 and _X5 are each independently selected from A, C, G, or U. In some embodiments, _X3 and/or _X5 are A. In some embodiments, _X3 and/or _X5 are C. In some embodiments, _X3 and/or _X5 are G. In some embodiments, _X3 and/or _X5 are U. In some embodiments, _Y4 is C. In other embodiments, _Y4 is not C. In some embodiments, _Y4 is A. In some embodiments, _Y4 is G. In other embodiments, _Y4 is not G. In some embodiments, Y4 is U.

In some embodiments, the cap-proximal sequence comprises a sequence that includes _N1 and _N2 of the cap structure, and _A3C4A5 (SEQ ID NO: 310). In some embodiments, _N1 and _N2 are each independently selected from A, _C , _G , or U. In some embodiments, _N1 is A and _N2 is G.

In some embodiments, the cap-proximal sequence comprises a sequence that includes _N1 and _N2 of the cap structure _{and A3U4G5} (SEQ ID NO:311). In some embodiments, _N1 and _N2 are each independently selected from A, _C , G, or U. In some embodiments, _N1 is A and _N2 is G.

Exemplary 5'UTRs include human alpha globin (hAg) 5'UTR or a fragment thereof, TEV 5'UTR or a fragment thereof, HSP70 5'UTR or a fragment thereof, or c-Jun 5'UTR or a fragment thereof.

In some embodiments, the RNA disclosed herein comprises a hAg 5'UTR or a fragment thereof. In some embodiments, the RNA disclosed herein comprises a hAg 5'UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the human alpha globin 5'UTR provided in SEQ ID NO: 312. In some embodiments, the RNA disclosed herein comprises a hAg 5'UTR provided in SEQ ID NO: 312.
SEQ ID NO:312
AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAGAACCC

In some aspects, the RNA disclosed herein comprises a hAg 5'UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the human alpha globin 5'UTR provided in SEQ ID NO: 313. In some aspects, the RNA disclosed herein comprises a hAg 5'UTR provided in SEQ ID NO: 313.
SEQ ID NO:313
AAACUAGUAUUCUUCUGGUCCCCAGAGACUCAGAGAGAACCC

In one embodiment, the DNA encoding the 5'UTR disclosed herein comprises a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to SEQ ID NO: 280. In one embodiment, the DNA encoding the 5'UTR comprises the sequence of SEQ ID NO:280. In one embodiment, the RNA disclosed herein comprises a 5'UTR comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to the 5'UTR provided in any of SEQ ID NOs: 281-282, with the transcribed 5'cap structure underlined. In one embodiment, the 5'UTR comprises any of SEQ ID NOs: 281-282, with the transcribed 5'cap structure underlined.
SEQ ID NO: 280 (DNA)
AG AATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC
SEQ ID NO: 281 (RNA)
AG AAUAAACUAGUAUUCUUCUGGUCCCACAGACUCAGAGAGAACCCGCCACC
SEQ ID NO: 282 (RNA)
AG AAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCACAGACΨCAGAGAGAACCCGCCACC

ii. 3′UTR
In some embodiments, the RNA disclosed herein comprises a 3'UTR. If present, the 3'UTR is located downstream of the protein coding sequence open reading frame, e.g., downstream of the stop codon of the protein coding region. The 3'UTR is typically the portion of the mRNA located between the protein coding sequence and the polyA tail of the mRNA. Thus, in some embodiments, the 3'UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence. The 3'UTR may be involved in regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.

The 3'UTR may also contain elements that are not encoded in the template from which the RNA is transcribed, but are added during post-transcriptional maturation, such as a polyA tail. The 3'UTR of an mRNA is not translated into an amino acid sequence. In some embodiments, the RNA disclosed herein comprises a 3'UTR that includes an F element and/or an I element. In some embodiments, the 3'UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an Xhol site.

In some aspects, the RNA disclosed herein comprises a 3'UTR having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or a percentage between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to the 3'UTR provided in SEQ ID NO: 314. In some aspects, the RNA disclosed herein comprises a 3'UTR provided in SEQ ID NO: 314.
SEQ ID NO:314
CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACCUCUC GUUCCAGACACCUCCCCAA GCACGCAGCAAUGCAGCUCAAACGCUUAGCCUAGCCACACCCCACGGGAAACAGCAGUGAUUAACCUUUA UUUCGUGCCAGCCACACC

In one embodiment, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to SEQ ID NO: 283. In one embodiment, the DNA encoding the 3'UTR comprises the sequence of SEQ ID NO:283. In one embodiment, the RNA disclosed herein comprises a 3'UTR comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to the 3'UTR provided in any of SEQ ID NOs: 284-285 and 317-318. In one embodiment, the 3'UTR comprises the sequence of any of SEQ ID NOs: 284-285 and 317-318.
SEQ ID NO: 283 (DNA)
CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCT CTGCTAGTTCCAGACACCTCCCCAAGC ACGCAGCAATGCAGCTCAAACGCTTAGCCTAGCCACACCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAAACGAAAGTTTAACTAAGCTATAACTAACCCCAGGGTTGGTCAATT TCGTGCCAGCCACACCCTGGAGCTAGC
SEQ ID NO: 284 (RNA)
CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCCUCCACCUGCCCCCACUCACCACCU CUGCUAGUUCCAGACACCUCCCCAAGC ACGCAGCAAUGCAGCUCAAACGCUUAGCCUAGCCACACCCCACGGGAAACAGCAGUGAUUAACCUUUA UCGUGCCAGCCACACCCUGGAGCUAGC
SEQ ID NO: 285 (RNA)
CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨ CCACCΨGCCCACCΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGC ACGCAGCAAΨGCAGCΨCAAAACGCΨΨAGCCΨAGCCACACCCCACGGGAAAACA ACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACCCΨGGAGCΨAGC
SEQ ID NO: 317 (RNA)
CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCCUCCACCUGCCCCCACUCACCACCU CUGCUAGUUCCAGACACCUCC CAAGCACGCAGCAAUGCAGCUCAAACGCUUAGCCUAGCCACACCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGUUGGU CAAUUUCGUGCCAGCCACACC
SEQ ID NO: 318 (RNA)
CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨ CCACCΨGCCCACACΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCC CAAGCACGCAGCAAΨGCAGCΨCAAAACGCΨΨAGCCΨAGCCACACCCCCCGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨΨAACΨAAGCΨAΨ ACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACC

D. Open Reading Frame (ORF)
The 5' and 3' UTRs may be operably linked to an open reading frame (ORF), which may be a sequence of codons capable of being translated into a polypeptide of interest. An open reading frame may be a sequence of several DNA or RNA nucleotide triplets that can be translated into a peptide or protein. An ORF may begin with an initiation codon at its 5' end, such as a combination of three consecutive nucleotides (ATG or AUG) that usually encodes the amino acid methionine, as well as a subsequent region that usually has a length of multiple three nucleotides. An open reading frame may end with at least one stop codon, including but not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some aspects, the open reading frame may end with one, two, three, four or more stop codons, including, but not limited to, TAATAA (SEQ ID NO:289), TAATAG (SEQ ID NO:290), TAATGA (SEQ ID NO:291), TAGTGA (SEQ ID NO:292), TAGTAA (SEQ ID NO:293), TAGTAG (SEQ ID NO:294), TGATGA (SEQ ID NO:295), TGATAG (SEQ ID NO:296), TGATAA (SEQ ID NO:297), or UAAUAA (SEQ ID NO:298), UAAUAG (SEQ ID NO:299), UAAUGA (SEQ ID NO:300), UAGUGA (SEQ ID NO:301), UAGUAA (SEQ ID NO:302), UAGUAG (SEQ ID NO:303), UGAUGA (SEQ ID NO:304), UGAUAG (SEQ ID NO:305), UGAUAA (SEQ ID NO:306), or any combination thereof. An open reading frame may be isolated or may be incorporated into a longer nucleic acid sequence, such as a vector or mRNA. An open reading frame may also be referred to as a "(protein) coding region" or "coding sequence."

As described herein, an RNA molecule may contain one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames.

In some embodiments, the ORF encodes a nonstructural viral gene. In some embodiments, the ORF further comprises one or more subgenomic promoters. In some embodiments, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some embodiments, the first RNA molecule does not comprise an ORF encoding any polypeptide of interest, while the second RNA molecule comprises an ORF encoding a polypeptide of interest. In some embodiments, the first RNA molecule does not comprise a subgenomic promoter.

The present disclosure provides an RNA molecule comprising at least one open reading frame encoding a Varicella-Zoster Virus (VZV) polypeptide. In some embodiments, the RNA molecule comprises at least one open reading frame encoding a VZV gE polypeptide.

E. Gene of interest The RNA molecule described herein may comprise 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, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties, polypeptides encoded by the human genome that have not yet been identified as therapeutic indications, but that nevertheless have utility in the fields of research and drug discovery, or combinations thereof. The sequence of a particular gene of interest can be easily identified by those skilled in the art using public and private databases, such as GENBANK®.

In some embodiments, the RNA molecule comprises a coding region for a gene of interest. In some embodiments, the gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or fragment thereof. In some embodiments, the antigenic polypeptide comprises one epitope from the antigen. In some embodiments, the antigenic polypeptide comprises multiple distinct epitopes from the antigen. In some embodiments, the antigenic polypeptide comprising multiple distinct epitopes from the antigen is a polyepitope. 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 autoantigen polypeptide.

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

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

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

In some embodiments, the RNA molecule encodes a VZV gE protein or a fragment or variant thereof. In some embodiments, the RNA molecule comprises a VZV polypeptide comprising an amino acid sequence according to any one of GENBANK® Accession Nos.: AAG32558.1, ABE03086.1, AAK01047.1, Q9J3M8.1, AEW88548.1, AGY33616.1, AEW89124.1, AIT53150.1, CAA25033.1, NP_040190.1, AKG56356.1, AEW89412.1, ABF21714.1, ABF21714.1, AAT07749.1, AEW88764.1, AAG48520.1, and/or AEW88980.1 (each of which sequences are incorporated herein by reference). In some embodiments, the RNA molecule encodes a VZV gE protein comprising an amino acid sequence according to GENBANK® Accession No. AH009994.2, which sequence is incorporated herein by reference.

In some embodiments, the RNA polynucleotide described herein or the composition or pharmaceutical preparation comprising the same comprises a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence having at least 80% identity to a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some embodiments, the RNA polynucleotide described herein or the composition or pharmaceutical preparation comprising the same is transcribed by a DNA template. In some embodiments, the DNA template used to transcribe 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 an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide encodes a polypeptide having at least 80% identity to a polypeptide sequence disclosed herein. In some embodiments, the polypeptide described herein is encoded by an RNA polynucleotide transcribed by a DNA template comprising a sequence complementary to the RNA polynucleotide.

In some embodiments, the RNA molecule encodes a VZV glycoprotein comprising any one of the sequences of SEQ ID NOs: 1-11, or a fragment or variant thereof.

In some embodiments, the RNA molecule encodes a VZV glycoprotein, or a fragment or variant thereof, synthesized from a nucleic acid sequence comprising any one of SEQ ID NOs: 12-145.

F. Poly A Tail In some embodiments, the RNA molecules disclosed herein include a polyadenylate (poly A) sequence, e.g., as described herein. In some embodiments, the poly A sequence is located downstream of the 3'UTR, e.g., adjacent to the 3'UTR. A "poly A tail" or "poly A sequence" refers to a stretch of consecutive adenine residues that may be attached to the 3' end of an RNA molecule. Poly A sequences are known to those of skill in the art and may follow the 3'UTR in the RNA molecules described herein. The poly A tail may increase the half-life of the RNA molecule.

The RNA molecules disclosed herein may have a polyA sequence that is attached to the free 3' end of the RNA after transcription by a template-independent RNA polymerase, or that is encoded by DNA and transcribed by a template-dependent RNA polymerase. In some embodiments, the polyA sequence is attached during RNA transcription, e.g., during preparation of the transcribed RNA in vitro, based on a DNA template that contains repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.

A DNA sequence that encodes a polyA sequence (the coding strand) 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 interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences may be at least 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, up to 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, exactly 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, or 5, 6, 7, 8, 9, 10, 11, 1 The length may be between any two of 2, 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 nucleotides. Such cassettes are disclosed in WO 2016/005324 A1 and are incorporated herein by reference. Any polyA cassette disclosed in WO 2016/005324 A1 may be used in the present invention. PolyA cassettes consisting essentially of dA nucleotides, but interrupted by random sequences with equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5-50 nucleotides, exhibit, at the DNA level, consistent propagation of plasmid DNA in E. coli, and at the RNA level, are still associated with beneficial properties for supporting RNA stability and translation efficiency, and are also encompassed. In some aspects, the polyA sequences contained in the RNA polynucleotides described herein consist essentially of adenosine nucleotides, but are interrupted by random sequences of the four nucleotides (A, C, G, U). Such random sequences may include at least 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, up to 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, exactly 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, or 5, 6, 7, 8, 9, 10, 11, 1 It may be any number of nucleotides in length between any two of 2, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

In some embodiments, no nucleotides other than adenosine nucleotides are adjacent to the polyA sequence at its 3' end, e.g., the polyA sequence is not masked or followed by nucleotides other than adenosine at its 3' end.

In some embodiments, the RNA molecule may further comprise an endonuclease recognition site sequence immediately downstream of the poly A tail sequence. The RNA molecule may further comprise a poly A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

The polyA sequence may be of any length. In some embodiments, the polyA tail may comprise a length of 5 to 300 nucleotides. In some embodiments, the RNA molecule comprises a polyA tail that comprises, consists essentially of, or consists of a sequence of about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the poly A tail is at least 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, or 50 0, up to 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, or 500, or exactly 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, 2 80, 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, or 500 pieces, or 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, 1 85, 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, or 500 adenosine nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the polyA 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%, by number of nucleotides in the polyA sequence, are adenosine nucleotides, but allows for the remaining nucleotides to be nucleotides other than adenosine nucleotides, e.g., uridine, guanosine, or cytosine. In this context, "consisting of" means that all nucleotides in the polyA sequence, e.g., 100% by number of nucleotides in the polyA sequence, are adenosine nucleotides.

In some embodiments, the RNA molecule comprises a poly-A tail that comprises a sequence of more than 30 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly-A tail that comprises about 40 adenosine nucleotides. In some embodiments, the RNA molecule comprises a poly-A tail that comprises about 80 adenosine nucleotides. In some embodiments, the 3' poly-A tail has a stretch of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some specific embodiments, the RNA molecule comprises about 40 consecutive adenosine residues. In some embodiments, the RNA molecule comprises about 80 consecutive adenosine residues. The poly-A tail may play a key regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may be a signal for RNA degradation. Some designs include a poly-A tail of about 40 adenosine nucleotides per adenosine nucleotide.

In some embodiments, a polyA tail may be located within an RNA molecule or other nucleic acid molecule, e.g., in a vector, e.g., in a vector that serves as, e.g., a template, for the generation of an RNA, e.g., an mRNA, by transcription of the vector. In some embodiments, an RNA molecule may not include a polyA tail.

In one embodiment, the DNA encoding the poly A tail disclosed herein comprises a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to SEQ ID NO: 286. In one embodiment, the DNA encoding the poly A tail comprises the sequence of SEQ ID NO: 286. In one embodiment, the RNA disclosed herein comprises a poly-A tail comprising a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, up to 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, exactly 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, or between any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%, to any of SEQ ID NOs: 287-288 and 315-316. In one embodiment, the poly-A tail comprises a sequence of any of SEQ ID NOs: 287-288 +/- 2 adenosine (A) nucleotides. In one embodiment, the poly A tail comprises the sequence of any of SEQ ID NOs: 287-288 +/- 1 adenosine (A) nucleotides. In one embodiment, the poly A tail comprises the sequence of any of SEQ ID NOs: 287-288. In one embodiment, the poly A tail comprises the sequence of any of SEQ ID NOs: 315-316 +/- 2 adenosine (A) nucleotides. In one embodiment, the poly A tail comprises the sequence of any of SEQ ID NOs: 315-316 +/- 1 ....
SEQ ID NO: 286 (DNA)
AAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO: 287 (RNA)
AAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO: 288 (RNA)
AAAAAAAAAAAAAAAAAAAAAAAAAGCAΨAΨGACΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO: 315 (RNA)
AAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
SEQ ID NO: 316 (RNA)
AAAAAAAAAAAAAAAAAAAAAAAAAGCAΨAΨGACΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

G. Self-amplifying RNA (SARNA)
In some embodiments, the RNA molecule may be saRNA. "Self-amplifying RNA", "self-amplifying RNA", and "replicon" refer to RNA that has the ability to replicate by itself. Self-amplifying RNA molecules may be produced by using replication elements, e.g., from alphaviruses, and replacing structural viral polypeptides with nucleotide sequences that encode a polypeptide of interest. Self-amplifying RNA molecules are typically positive-stranded molecules that can be directly translated after delivery to a cell, which translation provides an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNA molecules. In addition to these daughter RNA molecules, the collinear subgenomic transcripts may themselves be translated to provide in situ expression of the encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide additional transcripts having the same sense as the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this sequence of transcription is an amplification in the number of saRNA molecules introduced, such that the encoded gene of interest, e.g., a viral antigen, becomes the major polypeptide product of the cell.

In some embodiments, the self-amplifying RNA includes at least one or more genes including any one or combination of viral replicase, viral protease, viral helicase, and other nonstructural viral proteins. In some embodiments, the self-amplifying RNA may also include 5' and 3' terminal tractive replicative sequences, and optionally a heterologous sequence encoding a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter directing expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).

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

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

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

In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen, or, optionally, two or more polypeptide antigens linked together (e.g., linked in series) such that each of the sequences retains its identity when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as fusion polypeptides or may be engineered in a manner that results in separate polypeptide or peptide sequences.

In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a broad range of epitopes. In some embodiments, the self-amplifying RNA described herein may encode epitopes that have the ability to elicit either a helper T cell response or a cytotoxic T cell response, or both.

IV. RNA Transcription In some embodiments, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of this disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. The RNA may then be translated into a peptide or protein.

According to the present disclosure, "transcription" includes "in vitro transcription" or "IVT", which refers to a process in which transcription occurs in a non-cellular system in vitro to produce a synthetic RNA product for use in various applications, including, for example, the production of a protein or polypeptide. Cloning vectors may be applied for the generation of the transcript. These cloning vectors are commonly referred to as transcription vectors and are encompassed by the term "vector" according to the present invention. According to a specific embodiment, the RNA used is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription may be any promoter for any RNA polymerase. Particular 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. A DNA template for in vitro transcription may be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA may be obtained by reverse transcription of RNA.

Synthetic IVT RNA products may be translated in vitro or directly introduced into a cell and translated in the cell. With respect to RNA, the terms "expression" or "translation" refer to the process in the ribosomes of a cell in which a chain of mRNA directs the assembly of a sequence of amino acids to make 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, ribozymes, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, and small nucleolar RNA molecules. IVT reactions typically utilize a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified or modified ribonucleotide triphosphates), and a suitable RNA polymerase as described and/or utilized herein.

In some embodiments, mRNA is produced 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 may be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling the transcription may be any promoter for any RNA polymerase. The DNA template for in vitro transcription may be obtained by cloning a nucleic acid, in particular a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

In some embodiments, starting materials for IVT may include a linearized DNA template, nucleotides, an RNase inhibitor, pyrophosphatase, and/or T7 RNA polymerase. In some embodiments, the IVT process is carried out in a bioreactor. The bioreactor may include a mixer. In some embodiments, nucleotides may be added in the bioreactor throughout the IVT process.

In some embodiments, one or more post-IVT additives are added to the IVT mixture including RNA in the bioreactor after the IVT process. Exemplary post-IVT additives may include DNAse I configured to digest the linearized DNA template, and proteinase K configured to digest DNAse I and T7 RNA polymerase. In some embodiments, the post-IVT additives are incubated with the mixture in the bioreactor after the IVT process. In some embodiments, the bioreactor comprises at least 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, up to 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, exactly 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, or 60, 70, 80, 90, 100, 110, The IVT mixture may contain between any two or more of 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. The IVT mixture may be at least 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. Large at 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, and exactly at 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, or 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 of RNA or greater.

In some embodiments, the IVT mixture may include residual spermidine, residual DNA, residual proteins, peptides, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+, Na+, Ca2+), RNA fragments, residual nucleotides, free phosphates, or any combination thereof.

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

In some embodiments, both "ultrafiltration" and "diafiltration" refer to membrane filtration processes. Ultrafiltration is performed to a particle size of at least 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, up to 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, and ... Typically, membranes having a pore size between any two of 0.02, 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 are used. In some embodiments, ultrafiltration membranes are typically classified by molecular weight cut-off (MWCO) rather than pore size. For example, the MWCO may be at least 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, 410 kDa, 420 kDa, 430 kDa, 440 kDa, 450 kDa, 460 kDa, 470 kDa, 480 kDa, 490 kDa, 500 kDa, 510 kDa, 520 kDa, 530 kDa, 540 kDa, 550 kDa, 560 kDa, 570 kDa, 580 kDa, 590 kDa, 600 kDa, 610 kDa, 620 kDa, 630 kDa, 640 kDa, 650 kDa, 66 Da, 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 1000 0 kDa, up to 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 Da, 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, precisely 21 0kDa, 220kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa a, 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, or 30 kDa, 40 kDa, 50kDa, 60kDa, 70kDa, 80kDa, 90kDa, 100kDa, 110kDa, 120kDa, 130kDa, 140kDa, 150kDa, 160kDa, 170kDa, 180kDa, 190kDa, 200kDa, 210kDa, 220 kDa, 230kDa, 240kDa, 250kDa, 260kDa, 270kDa, 280kDa, 290kDa, 300kDa, 310kDa, 320kDa , 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 between any two of the above. Those skilled in the art will appreciate that the filtration membrane may be of different suitable materials, including, for example, polymers, cellulose, ceramics, etc., depending on the application. In some embodiments, membrane filtration may be more desirable for large volume purification processes.

In some embodiments, ultrafiltration and diafiltration of an IVT mixture to purify RNA may include (1) direct flow filtration (DFF), also known as "dead-end" filtration, in which the feed stream is applied perpendicular to the membrane plane and attempts to pass 100% of the fluid through the membrane, and/or (2) tangential flow filtration (TFF), also known as cross-flow filtration, in which the feed stream passes parallel to the membrane plane, with one portion passing through the membrane (permeate) while the remaining portion (retentate) is retained and/or recycled to the feed tank.

In some embodiments, filtration of the IVT mixture is performed via TFF, including an ultrafiltration step, a first diafiltration step, and a second diafiltration step. In some embodiments, the first diafiltration step is performed in the presence of ammonium sulfate. The first diafiltration step may be configured to remove a majority of impurities from the IVT mixture. In some embodiments, the second diafiltration step is performed without ammonium sulfate. The second diafiltration step may be configured to move the RNA into the DS buffer formulation.

A filtration membrane with an appropriate MWCO may 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 are retained in the retentate. The MWCO of the TFF membrane may be selected such that substantially all of the solutes of interest (e.g., the desired synthesized RNA species) remain in the retentate while undesired components (e.g., excess ribonucleotides, small nucleic acid fragments, e.g., digested or hydrolyzed DNA templates, peptide fragments, e.g., digested proteins and/or other impurities) pass through into the filtrate. In some embodiments, the retentate containing the desired synthesized RNA species may be recycled to the feed reservoir and refiltered in additional cycles. In some embodiments, the TFF membrane may have a MWCO of at least 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, up to 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, exactly 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or between any two of 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or greater. In some embodiments, the TFF membrane may have a MWCO of at least 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, up to 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, exactly 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or between any two of 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa or greater. In some embodiments, the TFF membrane may have a MWCO of about 250-350 kDa. In some embodiments, the TFF membrane (e.g., a cellulose-based membrane) may have a MWCO of about 30-300 kDa; in some embodiments, about 50-300 kDa, about 100-300 kDa, or about 200-300 kDa.

Diafiltration may be performed discontinuously or, alternatively, continuously. For example, in continuous diafiltration, diafiltration solution may be added to the sample feed reservoir at the same rate as filtrate is produced. In this way, the volume in the sample reservoir remains constant, but small molecules (e.g., salts, solvents, etc.) that can freely permeate 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 (e.g., salts, solvents, etc.) remaining in the reservoir is reached. Each additional diafiltration volume (DV) further reduces the small molecule (e.g., solvent) concentration. Continuous diafiltration typically requires a minimum volume for a given reduction in the molecules being filtered. Discontinuous diafiltration, on the other hand, allows for rapid changes in retentate conditions, e.g., pH, salt content, etc. In some embodiments, the first diafiltration step is carried out for diavolumes equal to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, up to 2, 3, 4, 5, 6, 7, 8, 9, 10, exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, or any two of 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater. In some embodiments, the second diafiltration step is performed with a diavolume equal to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, at most 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or between or greater than any two of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. In some embodiments, the first diafiltration step is performed with 5 diavolumes and the second diafiltration step is performed with 10 diavolumes.

In some embodiments, for ultrafiltration and/or diafiltration, the IVT mixture is at least 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, 70 0, 800, 900, or 1000 L/m2, up to 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/m2, exactly 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/m2, or 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/m2, or a rate equal to the greater L/m2 of filter area per hour. The concentrated RNA solution may contain at least 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL, up to 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL, exactly 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL, or between any two of 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL of single stranded RNA.

The bioburden of the RNA solution concentrated via filtration to obtain an RNA product solution may also be reduced in some embodiments. Filtration to reduce bioburden may be performed using one or more filters. The one or more filters may include filters having a pore size of at least 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, up to 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, exactly 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or between any two of 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.

As one example, the reduction of bioburden may include draining a retentate tank containing the retentate obtained from ultrafiltration and/or diafiltration to obtain a retentate. The reduction of bioburden may include flushing a filtration system for ultrafiltration and/or diafiltration using a wash buffer solution to obtain a wash pool solution that includes residual RNA remaining in the filtration system. The retentate may be filtered to obtain a filtered retentate. The wash pool solution may be filtered using a first 0.2 μm filter to obtain a filtered wash pool solution. The retentate may be filtered using the first 0.2 μm filter or another 0.2 μm filter.

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

V. RNA Encapsulation The RNA in the RNA product solution may be encapsulated, and the RNA solution may further comprise at least one encapsulating agent. In one embodiment, the encapsulating agent comprises lipids, lipid nanoparticles (LNPs), lipoplexes, polymer particles, polyplexes, and monolithic delivery systems, and combinations thereof.

In one embodiment, the encapsulating agent is a lipid, and lipid nanoparticle (LNP)-encapsulated RNA is produced. Without intending to be bound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and/or cationic polymers combine with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles. The lipids may be naturally occurring lipids or synthetic lipids. However, lipids are usually biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glucolipids, sulfatides, lipids with ether- and ester-linked fatty acids and polymerizable lipids, and combinations thereof. A lipid is a substance that is insoluble in water and extractable with an organic solvent. 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 disclosure. The lipid component and the non-lipid may be attached to one another either covalently or non-covalently.

In some embodiments, LNPs may be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular RNases and/or engineered for systemic delivery of RNA to target cells. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecule is administered intravenously to a subject in need thereof. In some embodiments, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecule is administered intramuscularly to a subject in need thereof.

In one embodiment, the RNA in the RNA solution is at a concentration of less than 1 mg/mL. In another embodiment, the RNA is at a concentration of at least about 0.05 mg/mL. In another embodiment, the RNA is at a concentration of at least about 0.5 mg/mL. In another embodiment, the RNA is at a concentration of at least about 1 mg/mL. In another embodiment, the RNA concentration is from about 0.05 mg/mL to about 0.5 mg/mL. In another embodiment, the RNA is at a concentration of at least 10 mg/mL. In another embodiment, the RNA is at a concentration of at least 50 mg/mL. In some embodiments, the RNA is at least 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, up to 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 exactly 0. 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 between any two of about 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 a higher concentration.

The present disclosure provides RNA solutions and lipid preparation mixtures or compositions thereof, comprising at least one RNA, e.g., encoding an antigen (e.g., a VZV polypeptide), complexed with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipoplexes, and/or nanoliposomes. In some embodiments, the composition comprises lipid nanoparticles.

Lipid nanoparticles or LNPs refer to any form of particle produced when cationic lipids and optionally one or more additional lipids are combined, e.g., in an aqueous environment and/or in the presence of RNA. In some embodiments, the lipid nanoparticles are included in a formulation that may be used to deliver an active or therapeutic agent, e.g., a nucleic acid (e.g., mRNA), to a target site of interest (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, the lipid nanoparticles of the present disclosure include a nucleic acid. Such lipid nanoparticles typically include a cationic lipid and one or more excipients, e.g., one or more neutral lipids, charged lipids, steroids, lipids conjugated to a polymer, or combinations thereof. In some embodiments, the active or therapeutic agent, e.g., a nucleic acid (e.g., mRNA), may be encapsulated in the lipid portion of the lipid nanoparticle or in the aqueous space surrounded by part or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism's or cellular machinery, e.g., a deleterious immune response. The nucleic acid (e.g., mRNA) or portions thereof may also be associated and complexed with the lipid nanoparticle. Lipid nanoparticles may include any lipid capable of forming a particle to which a nucleic acid can be attached or in which one or more nucleic acids are encapsulated.

In some embodiments, the RNA molecules provided (e.g., saRNA, mRNA) may be formulated with LNPs. In some embodiments, the lipid nanoparticles may have an average diameter of about 1 to 500 nm. In some embodiments, the lipid nanoparticles may have an average diameter of 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 at least 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 70 nm, 80 nm, 90 nm, 90 nm, 100 nm, 100 nm, 150 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 15 ... 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, up to 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, or exactly 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, 1 45 nm, or 150 nm, or between any two of 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 are substantially non-toxic. The term "mean diameter" refers to the average hydrodynamic diameter of the particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which results in the so-called Z-average, which has the dimension of length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). Herein, the "mean diameter", "diameter", or "size" of a particle is used synonymously with this value of the Z-average.

The LNPs described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the LNP may be at least 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, up to 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, exactly 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, or 0.1, 0.11, 0.1 2, 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. The polydispersity index is calculated in some embodiments based on dynamic light scattering measurements by the so-called cumulant analysis mentioned in the definition of "average diameter". Under certain conditions, it may be taken as an indication of the size distribution of an ensemble of nanoparticles.

In certain embodiments, the nucleic acid (e.g., RNA molecule) is resistant in aqueous solution to degradation by nucleases when present in the provided LNPs. In some embodiments, the LNPs are liver-targeting lipid nanoparticles. In some embodiments, the LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic LNPs may comprise at least one cationic lipid, at least one lipid conjugated to a polymer, and at least one helper lipid (e.g., at least one neutral lipid).

In certain embodiments, the RNA solution and its lipid preparation mixture or composition may be 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of a particular lipid, lipid type, or non-lipid component, such as a lipid-like material and/or a cationic polymer or an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid, or other material as disclosed herein or known to one of skill in the art, or may have at least 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 3 0%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, up to about 1%, about 2%, about 3%, about 4% About 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 3 0%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, exactly 1%, about 2%, about 3%, about 4% About 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30% %, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, or about 1%, about 2%, about 3%, about 4% About 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, 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%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, 6%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%. The specific lipids, lipid types, or non-lipid components, such as lipid-like materials and/or cationic polymers or adjuvants, antigens, peptides, polypeptides, sugars, nucleic acids, or other materials as disclosed herein or known to those of skill in the art, may be between any two of these.

The LNPs described herein may be prepared using a wide variety of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer and mixing the colloid with nucleic acid to obtain nucleic acid particles. The term "colloid" as used herein refers to a type of homogenous mixture in which the dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic in size and have a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture and not the entire suspension.

For the preparation of colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer, methods conventionally used to prepare liposomal vesicles and appropriately adapted are applicable here. The most commonly used methods for preparing liposomal vesicles share the following basic stages: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydration of the dried lipids (using various aqueous media). In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried down to result in a thin film at the bottom of a flask. The resulting lipid film is hydrated using a suitable aqueous medium to produce a liposomal dispersion. Furthermore, an additional downsizing step may be included.

Reverse phase evaporation is an alternative method to film hydration for preparing liposomal vesicles that involves the formation of a water-in-oil emulsion between the aqueous phase and the lipid-containing organic phase. Brief sonication of the mixture is required to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel that subsequently becomes the liposomal suspension.

The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected through a needle into an aqueous solution. This action disperses the lipids throughout the solution and promotes lipid structure formation, e.g., lipid vesicle formation, e.g., liposome formation. In general, the RNA lipoplex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, such a colloidal liposome dispersion is formed in some embodiments as follows: an ethanol solution containing lipids, e.g., cationic lipids and additional lipids, is injected into an aqueous solution under stirring. In some embodiments, the RNA lipoplex particles described herein can be obtained without an extrusion step.

The term "extrude" or "extrusion" refers to the creation of particles with a fixed cross-sectional profile. In particular, it refers to the downsizing of particles in which the particles are forced through a filter with defined pores.

Other methods having organic solvent-free characteristics may also be used in accordance with the present disclosure to prepare colloids.

In some embodiments, RNA encapsulated in LNPs may be produced by rapid mixing of an RNA solution (e.g., an RNA product solution) as described herein and a lipid preparation (e.g., comprising at least one cationic lipid and optionally one or more other lipid components in an organic solvent) as described herein under conditions such that a sudden change in the solubility of the lipid components is triggered, which drives the lipids towards self-assembly in the form of LNPs. In some embodiments, suitable buffering agents include tris, histidine, citrate, acetate, phosphate, or succinate. The pH of the liquid formulation is related to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidified buffer may be at least half a pH scale lower than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half a pH scale higher than the pKa of the encapsulating agent (e.g., cationic lipid). In some embodiments, the properties of the cationic lipids are selected such that de novo formation of the particles occurs by association with the oppositely charged backbone of the nucleic acid (e.g., RNA). In this way, the particles are formed around the nucleic acid, which, for example, in some embodiments, can result in much higher encapsulation efficiency than would be achieved in the absence of an interaction between the nucleic acid and at least one of the lipid components.

In certain embodiments, the nucleic acid, when present in the lipid nanoparticle, is resistant in aqueous solution to degradation by nucleases. Lipid nanoparticles containing nucleic acids and methods for their preparation are disclosed, for example, in U.S. Patent Application Publication Nos. 2004/0142025, 2007/0042031, and PCT Publication Nos. WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety and for all purposes.

Some aspects described herein relate to compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or more nucleic acid species, e.g., RNA species. In an LNP formulation, each nucleic acid species can be formulated separately as an individual LNP formulation. In that case, each individual LNP formulation contains one nucleic acid species. The individual LNP formulations may be present as separate entities, e.g., in separate containers. Such formulations can be obtained by providing each nucleic acid species separately (typically each in the form of a nucleic acid-containing solution) with suitable cationic or cationically ionizable lipid or lipid-like material and a cationic polymer that allows the formation of LNPs. Each particle exclusively contains the unique nucleic acid species that is provided when the particle is formed (individual particulate formulation).

In some embodiments, a composition, e.g., a pharmaceutical composition, comprises more than one individual LNP formulation. Each pharmaceutical composition is referred to as a mixed LNP formulation. A mixed LNP formulation according to the present invention may be obtained by forming the individual LNP formulations separately, as described above, and then mixing the individual LNP formulations. The mixing step may result in a formulation comprising a mixed population of nucleic acid-containing LNPs. The individual LNP populations may be together in one container, comprising a mixed population of individual LNP formulations.

Alternatively, different nucleic acid species can be formulated together as a combined LNP formulation. Such a formulation can be obtained by providing a combined formulation (typically a combined solution) of different RNA species together with suitable cationic or cationically ionizable lipid or lipid-like materials and cationic polymers that allow the formation of LNPs. In contrast to mixed LNP formulations, combined LNP formulations typically include LNPs that contain more than one RNA species. In a combined LNP composition, the different RNA species are typically present together in a single particle.

A. Cationic Polymeric Materials Given their high degree of chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups consist of amines that change state of protonation in the pH range of 5.5-7.5, which is believed to lead to ionic imbalances that often result in endosomal rupture. Besides polymers such as poly-L-lysine, polyamidoamines, protamines and polyethyleneimines, naturally occurring polymers such as chitosan have all been applied in nucleic acid delivery and are suitable as cationic materials useful in some embodiments herein. Additionally, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. Poly(P-amino esters) have gained widespread use in nucleic acid delivery, particularly due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials herein.

"Polymer material," as used herein, is given its ordinary meaning, e.g., a molecular structure that includes one or more repeating units (monomers) connected by covalent bonds. In some embodiments, such repeating units may all be identical; alternatively, in some cases, more than one type of repeating unit may be present in the polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer, e.g., a protein. In some cases, additional moieties may also be present in the polymeric material, e.g., a targeting moiety, e.g., a targeting moiety described herein.

Those skilled in the art will recognize that when more than one type of repeat unit is present within a polymer (or polymer portion), the polymer (or polymer portion) is said to be a "copolymer." In some embodiments, a polymer (or polymer portion) utilized in accordance with the present disclosure may be a copolymer. The repeat units forming the copolymer may be arranged in any manner. For example, in some embodiments, the repeat units may be arranged in a random order; alternatively or additionally, in some embodiments, the repeat units may be arranged in alternating order or as a "block" copolymer, including, for example, one or more regions (e.g., a first block) each including a first repeat unit and one or more regions (e.g., a second block) each including a second repeat unit. A block copolymer may have two (diblock copolymer), three (triblock copolymer), or a greater number of distinct blocks.

In certain embodiments, the polymeric materials for use according to the present disclosure are biocompatible. A biocompatible material is a material that does not typically result in significant cell death at reasonable concentrations. In certain embodiments, a biocompatible material is biodegradable, e.g., capable of degrading chemically and/or biologically within a physiological environment, e.g., within the body. In certain embodiments, the polymeric material may be or may include protamine or a polyalkyleneimine, particularly protamine.

As those skilled in the art will recognize, the term "protamine" is often used to refer to any of a variety of strongly basic proteins of relatively low molecular weight that are rich in arginine and are found in the sperm cells of various animals (e.g., fish) specifically in association with DNA instead of somatic histones. In particular, the term "protamine" is often used to refer to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and yields primarily arginine on hydrolysis. In purified form, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

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

In some embodiments, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine. In some embodiments, the polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the polyalkyleneimine is a linear polyalkyleneimine, e.g., linear polyethyleneimine (PEI).

Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those that can electrostatically bind to nucleic acids. In some embodiments, cationic polymeric materials contemplated for use herein include any cationic polymeric material with which nucleic acids can be associated, e.g., by forming a complex with the nucleic acid or by forming a vesicle in which the nucleic acid is surrounded or encapsulated.

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

B. Lipids & Lipid-Like Materials The terms "lipid" and "lipid-like materials" are used herein to refer to molecules that contain one or more hydrophobic moieties or groups, and optionally also contain one or more hydrophilic moieties or groups. According to the present disclosure, lipids and lipid-like materials may be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.

The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. In general, lipids can be divided into eight categories: fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids as well as sterol-containing metabolites such as cholesterol, and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramide sphingophospholipids (e.g., sphingomyelin, phosphocholine), and sphingoglycolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives.

The term "lipid-like material", "lipid-like compound", or "lipid-like molecule" refers to substances that are structurally and/or functionally related to lipids, but may not be considered lipids in the strict sense. For example, the term includes compounds that can form amphiphilic layers when present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment, and includes surfactants, or synthetic compounds that have both hydrophilic and hydrophobic portions. In general, the term refers to molecules that contain hydrophilic and hydrophobic portions with different structural organization that may or may not be similar to that of lipids.

In some embodiments, the RNA solution and its lipid preparation mixture or composition may include cationic lipids, neutral lipids, cholesterol, and/or lipids conjugated with a polymer (e.g., polyethylene glycol) that form lipid nanoparticles that encapsulate the RNA molecule. Thus, in some embodiments, the LNPs may include cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), lipids conjugated to a polymer (e.g., PEG lipids), or combinations thereof. In some embodiments, the LNPs include or encapsulate the nucleic acid molecule.

i. Cationic lipids Cationic or cationically ionizable lipids or lipid-like materials refer to lipids or lipid-like materials that have the ability to be positively charged and can electrostatically bind to nucleic acids. As used herein, "cationic lipids" or "cationic lipid-like materials" refer to lipids or lipid-like materials that have a net positive charge. Cationic lipids or lipid-like materials bind to negatively charged nucleic acids through electrostatic interactions. In general, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chain, and the head group of the lipid typically carries a positive charge. Exemplary cationic lipids include one or more amine groups that carry a positive charge. Cationic lipids may encapsulate negatively charged RNA.

In some embodiments, the cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on the pH. The ionization of the cationic lipids affects the surface charge of the lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, it is believed that this ionizable behavior enhances efficacy through aiding in endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH. For purposes of this disclosure, such "cationically ionizable" lipids or lipid-like materials are included in the terms "cationic lipids" or "cationic lipid-like materials" unless the context requires otherwise.

In some embodiments, the cationic lipid may comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipid present in the particle. In some embodiments, the cationic lipid comprises at least 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, up to 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, up to exactly 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, of the total lipid present in the particle. It may be 1%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, or any two of 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol%, or 100 mol%, or any range or value derivable therein.

Examples of cationic lipids are ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; Dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethyl dioleylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3- Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DM A), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2, 3-Bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (bAE-DMRIE) E), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2 -dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8, 8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl) oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N12-5), 1-[2-[bis(2-hydroxy Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102), but is not limited to: ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (Lipidoid 02-200); or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102).

In some embodiments, the lipid nanoparticles include one or more cationic lipids. In one embodiment, the lipid nanoparticles have the formula:

を有する（４－ヒドロキシブチル）アザンジイル）ビス（ヘキサン－６，１－ジイル）ビス（２－ヘキシルデカノエート）（ＡＬＣ－０３１５）を含む。陽イオン性脂質は、例えば、米国特許第１０，１６６，２９８号明細書において開示され、その全開示は参照により全体がすべての目的のために本明細書に組み込まれる。代表的な陽イオン性脂質は以下を含む：

Cationic lipids include (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), having the formula: Cationic lipids are disclosed, for example, in U.S. Pat. No. 10,166,298, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Representative cationic lipids include:

In some embodiments, the RNA-LNPs comprise a cationic lipid, an RNA molecule as described herein, and one or more of a neutral lipid, a steroid, a pegylated lipid, or a combination thereof. When more than one cationic lipid is incorporated within the LNP, such percentages apply to the combined cationic lipids. In one embodiment, the cationic lipids each comprise, for example, at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent, up to 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent. , or between any two of exactly 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent, or about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mole percent.

In some aspects of the present disclosure, the LNPs include a combination or mixture of any of the lipids described above.

ii. Lipids Conjugated to a Polymer In some embodiments, the LNPs comprise a lipid conjugated to a polymer. The term "lipid conjugated to a polymer" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a lipid conjugated to a polymer is a PEGylated lipid. The term "PEGylated lipid" refers to a molecule that comprises both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-s-DMG), 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and the like.

In certain embodiments, the LNPs include an additional stabilizing lipid that is a polyethylene glycol lipid (a PEGylated lipid). A polymer-conjugated lipid (e.g., a PEG lipid) refers to a molecule that includes both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a PEG lipid. A PEG lipid refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. PEG lipids include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Exemplary polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol lipid is N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol lipid is PEG-c-DOMG. In other embodiments, the LNPs are PEGylated diacylglycerols (PEG-DAGs), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAGs), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamates, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. PEG lipids are disclosed, for example, in U.S. Pat. No. 9,737,619, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, the lipid nanoparticles include a lipid conjugated to a polymer. In one embodiment, the lipid nanoparticles have the formula:

を有する、２－［（ポリエチレングリコール）－２０００］－Ｎ，Ｎ－ジテトラデシルアセトアミド（ＡＬＣ－０１５９）を含む。

The compound includes 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), which has the formula:

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

In certain embodiments, the PEG lipid is present in the LNP in an amount of about 1 to about 10 mole percent (mol%) (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%, exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol%) relative to the total lipid content of the nanoparticle.

iii. Additional Lipids In certain embodiments, the LNPs comprise one or more additional lipids or lipid-like materials that stabilize the formation of particles during their formation. Suitable stabilizing or structuring lipids include non-cationic lipids, such as neutral lipids and anionic lipids. Without being bound by any theory, optimization of the formulation of LNPs by the addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to ionizable/cationic lipids or lipid-like materials, may enhance particle stability and efficacy of nucleic acid delivery.

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

Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydro-sphingomyelin, cephalin, and cerebrosides. Exemplary phospholipids include, for example, phosphatidylcholine, for example, diacylphosphatidylcholine, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidoylphosphatidylcholine ... phatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycero-3-phosphocholine (OChemsPC). C16 lysoPC); and phosphatidylethanolamines, such as diacylphosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE-mal), dioleoylphosphatidylethanolamine (DOPE ... ), dimyristoylphosphoethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), distearoyl-phosphatidylethanolamine (DSPE), diphytanoyl-phosphatidylethanolamine (DpyPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-transPE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid has the formula:

を有する、１，２－ジステアロイル－ｓｎ－グリセロ－３ホスホコリン（ＤＳＰＣ）である。

and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), having the formula:

In some embodiments, the LNPs comprise a neutral lipid, and the neutral lipid comprises one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, or SM.

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

を含む化合物である。

It is a compound comprising:

In certain embodiments, the steroid or steroid analog is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In one embodiment, cholesterol has the formula:

を有する。

has.

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

In some embodiments, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol), may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipid present in the particle. In some embodiments, the non-cationic lipid, e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol), may comprise at least 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol%, up to 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol% of the total lipid present in the particle. It may be 1%, 70 mol%, 80 mol%, or 90 mol%, exactly 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol%, or between any two of 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol%.

VI. Characterization and Analysis of RNA Molecules The RNA molecules described herein may be analyzed and characterized using various methods. Analysis may be performed before or after capping. Alternatively, analysis may be performed before or after polyA capture-based affinity purification. In another embodiment, analysis may be performed before or after additional purification steps, such as anion exchange chromatography. For example, RNA template quality may be determined using a Bioanalyzer chip-based electrophoresis system. In other embodiments, RNA template purity is analyzed using analytical reverse-phase HPLC, respectively. Capping efficiency may be analyzed, for example, using total nuclease digestion followed by MS/MS quantification of dinucleotide capped species versus uncapped GTP species. In vitro efficacy may be analyzed, for example, by transfection of the RNA molecules into human cell lines. Protein expression of the polypeptide of interest may be quantified using methods, such as ELISA or flow cytometry. Immunogenicity may be analyzed, for example, by transfection of RNA molecules into cell lines, e.g., PBMCs, indicative of innate immune stimulation. Cytokine induction may be analyzed, for example, using methods, e.g., ELISA, to quantify cytokines, e.g., interferon-α. Biodistribution may be analyzed, for example, by bioluminescence measurements.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when assessed in an organism administered a composition or pharmaceutical preparation comprising the RNA polynucleotide, increased expression of a gene of interest (e.g., an antigen); increased duration of expression (e.g., longer expression) of the gene of interest (e.g., an antigen); increased expression and increased duration of expression (e.g., longer expression) of the gene of interest (e.g., an antigen); decreased interaction of the RNA polynucleotide with IFIT1; increased translation of the RNA polynucleotide is observed compared to a suitable reference.

In some embodiments, the reference includes an organism administered a similar RNA polynucleotide except that it does not have the m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2 cap. In some embodiments, the reference includes an organism administered a similar RNA polynucleotide except that it does not have the cap-proximal sequence disclosed herein. In some embodiments, the reference includes an organism administered a similar RNA polynucleotide except that it has a self-hybridizing sequence.

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

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

In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 2-fold to at least 10-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 2-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 3-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 4-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 6-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 8-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is at least 10-fold.

In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is about 2-fold to about 50-fold. In some embodiments, the increase in expression of the gene of interest (e.g., antigen) is about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold. In some embodiments, the increase in expression of a gene of interest (e.g., antigen) is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold , 48x, 49x, or 50x, up to 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, or 50x, exactly 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, or 50x, or ... 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, 26x, 27x, 28x, 29x, 30x, 31x, 32x, 33x, 34x, 35x, 36x, 37x, 38x, 39x, 40x, 41x, 42x, 43x, 44x, 45x, 46x, 47x, 48x, 49x, or 50x, or any range or value derivable therein.

In some embodiments, the increased expression (e.g., increased duration of expression) of the gene of interest (e.g., antigen) persists for at least 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours, up to 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours, exactly 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours, or any two of 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after administration of the composition or pharmaceutical preparation comprising the RNA polynucleotide. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 24 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 48 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 72 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 96 hours after administration. In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for at least 120 hours following administration of a composition or pharmaceutical preparation comprising the RNA polynucleotide.

In some embodiments, the increased expression of the gene of interest (e.g., antigen) persists for about 24-120 hours after administration of a composition or pharmaceutical preparation comprising an RNA polynucleotide. In some embodiments, the increased expression persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of a composition or pharmaceutical preparation comprising an RNA polynucleotide. In some embodiments, the increased expression of the gene of interest (e.g., antigen) lasts for at least 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, up to 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, exactly 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or between any two of 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or any range or value derivable therein.

VII. Immune Responses and Assays As discussed herein, the present disclosure relates to eliciting or inducing an immune response against a VZV protein, such as a wild-type or variant VZV glycoprotein, in a subject. In one aspect, the immune response can protect a subject from an infection or related disease, particularly an infection or related disease involving VZV, or can treat a subject who has, is suspected of having, or is at risk of developing an infection or related disease, particularly an infection or related disease involving VZV. One use of the immunogenic composition of the present disclosure is to prevent VZV infection by inoculating or vaccinating a subject.

A. Immunoassays The present disclosure includes the implementation of serological assays to assess whether and to what extent an immune response is induced or elicited by the compositions of the present disclosure. There are many types of immunoassays that may be implemented. Immunoassays encompassed by the present disclosure include, but are not limited to, those described in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (Western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo.

Immunoassays are generally binding assays. In some embodiments, immunoassays are various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. In one example, an antibody or antigen is immobilized on a selected surface, such as a well in a polystyrene microtiter plate, a dipstick, or a column support. A test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is then added to the well. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody may be detected. Detection is generally achieved by the addition of another antibody specific for the desired antigen or antibody, linked to a detectable label. This type of ELISA is known as a "sandwich ELISA." Detection may also be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.

Competitive ELISA is also a possible implementation, in which the test sample competes for binding with a known amount of labeled antigen or antibody. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with the coated well. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well, and therefore the final signal. Regardless of the format used, ELISAs have certain features in common, such as coating, incubation or binding, washing to remove non-specifically bound species, and detection of bound immune complexes.

The antigen or antibody may be linked to a solid support, for example in the form of a plate, bead, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, the wells of the plate are typically incubated with a solution of the antigen or antibody, either overnight or for a specified period of time. The wells of the plate are then washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with respect to the test antisera. These include solutions of bovine serum albumin (BSA), casein, and milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface, thus reducing the background caused by nonspecific binding of the antisera onto the surface.

B. Diagnosis of VZV Infection The present disclosure contemplates the use of VZV polypeptides, proteins, and/or peptides in a variety of ways, including detecting the presence of VZV to diagnose infection. According to the present disclosure, a method of detecting the presence of infection involves obtaining a sample, such as a sample taken from an individual suspected of being infected with one or more VZV strains, for example, from the individual's blood, saliva, tissue, bone, muscle, cartilage, or skin. After isolation of the sample, a diagnostic assay utilizing the polypeptides, proteins, and/or peptides of the present disclosure may be performed to detect the presence of VZV, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassays, Western blot analysis, and ELISA assays.

In general, the present disclosure contemplates a method of diagnosing an infectious disease in which a sample suspected of being infected with VZV is spiked with a polypeptide, protein, or peptide in accordance with the present disclosure, and VZV is indicated by binding of an antibody to the polypeptide, protein, and/or peptide, or binding of the polypeptide, protein, and/or peptide to an antibody in the sample.

Thus, RNA molecules encoding VZV polypeptides, proteins, and/or peptides according to the present disclosure may be used to treat, prevent, or reduce the severity of disease from infection resulting from VZV infection (e.g., active or passive immunization) or for use as research tools.

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

C. Protective Immunity In some aspects of the present disclosure, RNA molecules encoding VZV polypeptides, RNA-LNPs and compositions thereof confer protective immunity to a subject. Protective immunity refers to the body's ability to mount a specific immune response that protects the subject from developing a particular disease or condition involving an agent against which an immune response is mounted. An immunogenically effective amount is capable of conferring protective immunity to a subject.

As used herein, the phrase "immune response" or equivalently "immunological response" refers to the development of humoral (antibody-mediated), cellular (mediated by antigen-specific T cells or their secretory products), or both humoral and cellular responses directed against an antigen. Such responses may be active or passive. Cellular immune responses are elicited by the presentation of polypeptide epitopes in association with class I or class II MHC molecules to activate antigen-specific CD4(+) helper T cells and/or CD8(+) cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglial cells, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred in a subject from the production of antibodies in response to the presence of an antigen, e.g., a VZV polypeptide encoded by an RNA molecule of the present disclosure.

As used herein, "passive immunization" includes, but is not limited to, administration of activated immune effectors, including cellular or protein mediators of the immune response (e.g., monoclonal and/or polyclonal antibodies). Monoclonal or polyclonal antibody compositions may be used in passive immunization to treat, prevent, or reduce the severity of disease caused by infection with an organism bearing the antigen recognized by the antibody. The antibody composition may include antibodies that bind to a variety of antigens, and the antigens may be associated with a variety of organisms. The antibody component may be a polyclonal antiserum. In certain embodiments, one or more antibodies are affinity purified from an animal or second subject challenged with the antigen. Alternatively, an antibody mixture may be used that is a mixture of monoclonal and/or polyclonal antibodies against antigens present in the same, related, or different microorganisms or organisms, e.g., viruses, including but not limited to VZV.

Passive immunity may be conferred to a patient or subject by administering to the patient immunoglobulin (Ig) and/or other immune factors obtained from a donor or other non-patient source with known immune reactivity. In other aspects, the immunogenic compositions of the present disclosure may be administered to a subject who then acts as a source or donor of globulin ("hyperimmune globulin") produced in response to challenge with the immunogenic composition, containing antibodies directed against VZV or other organisms. The subject so treated donates plasma from which hyperimmune globulin is then obtained via conventional plasma fractionation methodologies and administered to another subject to confer resistance to or treat VZV infection.

For purposes of this specification and the appended claims, the terms "epitope" and "antigenic determinant" are used interchangeably to refer to a site on an antigen to which B and/or T cells respond or recognize. B cell epitopes may be formed both from contiguous amino acids or from non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically comprises at least 3, and more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols (1996). Antibodies that recognize the same epitope may be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. T cells recognize consecutive epitopes of about 9 amino acids for CD8 cells or about 13-15 amino acids for CD4 cells. T cells that recognize an epitope may be identified by in vitro assays that measure antigen-dependent proliferation as determined by ^3H -thymidine incorporation (Burke et al., 1994), antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996) or cytokine secretion by primed T cells in response to the epitope.

The presence of a cell-mediated immunological response may be determined by proliferation assays (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effect of an immunogenic composition may be distinguished by separately isolating IgG and T cells from an immunized syngeneic animal and measuring the protective or therapeutic effect in a second subject.

As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's or recipient's immune response, including IgG, IgD, IgE, IgA, IgM and related proteins. Under normal physiological conditions, antibodies are found in plasma and other body fluids and in the membranes of certain cells and are produced by a type of lymphocyte designated as a B cell or their functional equivalent.

As used herein, the terms "immunogenic agent" or "immunogen" or "antigen" are used interchangeably to describe a molecule that has the ability to induce an immunological response against itself upon administration to a recipient, either alone, in combination with an adjuvant, or presented on a presentation vehicle.

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

In some embodiments, the RNA molecules and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition that may be formulated into a solid, semi-solid, liquid, lyophilized, frozen, or gaseous preparation. In some embodiments, the RNA molecules and/or RNA-LNPs disclosed herein may be administered in a pharmaceutical composition that may include a pharma- ceutically acceptable carrier and may optionally include one or more adjuvants, stabilizers, salts, buffers, preservatives, and optionally other therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein include one or more pharma- ceutically acceptable carriers, diluents, and/or excipients. In some embodiments, the pharmaceutical compositions do not include an adjuvant (e.g., are adjuvant-free).

Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

The term "diluent" refers to a diluent and/or thinning agent. Further, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and/or mixed medium. Examples of suitable diluents include ethanol, glycerol saline and water.

The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, into which an active component is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. A carrier, as used herein, may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's, lactated Ringer's, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, among others, biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxy-propylene copolymers. In some embodiments, the pharmaceutical compositions of the present disclosure include sodium chloride.

Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro, ed., 1985).

Pharmaceutical carriers, excipients or diluents may be selected with regard to the intended route of administration and standard pharmaceutical practice.

In some embodiments, the composition comprises an RNA molecule comprising an open reading frame encoding an immunogenic polypeptide. In some embodiments, the immunogenic polypeptide comprises a VZV antigen. In some embodiments, the VZV antigen is a VZV polypeptide. In some embodiments, the VZV polypeptide is a VZV glycoprotein (e.g., gK, gN, gC, gB, gH, gM, gL, gI, and gE) or a fragment or variant thereof. In some embodiments, the RNA molecule encodes a VZV gK polypeptide, the RNA molecule encodes a VZV gN polypeptide, the RNA molecule encodes a VZV gC polypeptide, the RNA molecule encodes a VZV gB polypeptide, the RNA molecule encodes a VZV gH polypeptide, the RNA molecule encodes a VZV gM polypeptide, the RNA molecule encodes a VZV gL polypeptide, the RNA molecule encodes a VZV gI polypeptide, and/or the RNA molecule encodes a VZV gE polypeptide. In one embodiment, the RNA molecule encodes a VZV gE polypeptide. In some embodiments, the VZV polypeptide comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or more) VZV polypeptides.

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

A. Immunogenic Compositions Comprising LNPs In some embodiments, pharmaceutical compositions comprise RNA molecules (e.g., polynucleotides) disclosed herein formulated with a lipid-based delivery system. Thus, in some embodiments, the composition comprises a lipid-based delivery system (e.g., LNPs) (e.g., lipid-based vaccines) that delivers nucleic acid molecules to the inside of cells, where the nucleic acid molecules can then replicate, inhibit protein expression of interest, and/or express the encoded polypeptide of interest. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some embodiments, the composition comprises at least one RNA molecule encoding a VZV polypeptide that is complexed with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids, forming a lipid nanoparticle (LNP), liposome, lipoplex, and/or nanoliposome. In some embodiments, the composition comprises a lipid nanoparticle. Thus, in certain aspects, the present disclosure relates to compositions comprising one or more lipids associated with a nucleic acid or a polypeptide/peptide (eg, VZV RNA-LNPs).

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

In further embodiments, the immunogenic composition comprising the lipid-based delivery system further comprises a stabilizing agent. In some embodiments, the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrrolidone, glycine, or a combination thereof. In some embodiments, the stabilizing agent is a disaccharide, or a sugar. In one embodiment, the stabilizing agent is sucrose. In another embodiment, the stabilizing agent is trehalose. In a further embodiment, the stabilizing agent is a combination of sucrose and trehalose. In some embodiments, the total concentration of the stabilizing agent in the composition is about 5% to about 10% w/v. For example, the total concentration of stabilizers may be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v, at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v, or exactly 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. w/v, or any range or value derivable therein between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v. In some embodiments, the stabilizing agent concentration includes, but is not limited to, a concentration of about 10 mg/mL to about 400 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 150 mg/mL, about 100 mg/mL to about 140 mg/mL, about 100 mg/mL to about 130 mg/mL, about 100 mg/mL to about 120 mg/mL, about 100 mg/mL to about 110 mg/mL, or about 100 mg/mL to about 105 mg/mL. In some embodiments, the concentration of the stabilizing agent is 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 500 mg/mL, 600 mg/mL, 700 mg/mL, 800 mg/mL, 900 mg/mL, 10 ... mL, at least 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, and up to 10mg/mL, 20mg/mL, 50mg/mL, 100mg/mL, 101mg/mL, 102mg/mL, 103mg/mL, 104mg/mL, 105mg/mL, 106mg/mL, 107mg/mL, 108mg/mL, 109mg/mL, 110mg/mL L, 150mg/mL, 200mg/mL, 300mg/mL, 400mg/mL, or 10mg/mL, 20m g/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL or between any two or a higher concentration.

In further embodiments, the mass of the stabilizer and the mass of the RNA are in a specific ratio. In one embodiment, the mass ratio of the stabilizer and the RNA is 5000 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 2000 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 1000 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 500 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 100 or less. In another embodiment, the mass ratio of the stabilizer and the pharmaceutical agent is 50 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 10 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 1 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 0.5 or less. In another embodiment, the mass ratio of the stabilizer and the RNA is 0.1 or less. In another embodiment, the stabilizer and the RNA constitute a mass ratio of about 200-2000 stabilizer:1 RNA.

In some embodiments, the immunogenic composition comprising a lipid-based delivery system further comprises a buffer. Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, Tris hydrochloride (HCl), amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and/or combinations thereof. In some embodiments, the buffer is HEPES buffer, Tris buffer, or PBS buffer. In one embodiment, the buffer is Tris buffer. In another embodiment, the buffer is HEPES buffer. In a further embodiment, the buffer is PBS buffer. For example, the buffer concentration is at least 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, and at most 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, and at most 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, and at most 1 mM, 2 mM, 3 mM, 4 mM, M, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or equal to any range or value derivable therein. The buffer may be at neutral pH, pH 6.5-8.5, pH 7.0-pH 8.0, or pH 7.2-pH 7.6. For example, the buffer may be at least pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, up to pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or exactly pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein between any two of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5. In a specific embodiment, the buffer is at pH 7.4.

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

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

In some embodiments, the immunogenic composition comprising the lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof. As used herein, "any other excipient" includes, but is not limited to, an antioxidant, glutathione, EDTA, methionine, desferal, an antioxidant, a metal scavenger, or a free radical scavenger. In one embodiment, the surfactant, preservative, excipient, or a combination thereof is sterile water for injection (sWFI), bacteriostatic water for injection (BWFI), saline, dextrose solution, polysorbate, poloxamer, Triton, a divalent cation, lactated Ringer's, an amino acid, a sugar, a polyol, a polymer, or a cyclodextrin.

Examples of excipients, which refer to ingredients in an immunogenic composition that are not active ingredients, include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants. Preservatives for use in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, "pharmaceutically acceptable carriers" includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluid and nutrient replenishers, similar materials as known to those of skill in the art, and combinations thereof. Diluents, or diluting or thinning agents, include, but are not limited to, ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, corn, rice, and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition may range from about 10% to about 90%, about 25% to about 75%, about 30% to about 60%, or about 12% to about 60% by weight of the total composition.

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

In one embodiment, the pharmaceutical composition comprises a VZV RNA molecule encoding a VZV polypeptide disclosed herein complexed with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids to form a VZV RNA-LNP. In some embodiments, the VZV RNA-LNP composition is liquid. In some embodiments, the VZV RNA-LNP composition is frozen. In some embodiments, the VZV RNA-LNP composition is lyophilized. In some embodiments, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide molecule encoding a VZV polypeptide disclosed herein encapsulated in a LNP having a lipid composition of cationic lipid, PEGylated lipid (i.e., PEG lipid), and one or more structured lipids (e.g., neutral lipid).

In some embodiments, the VZV RNA-LNP compositions comprise a cationic lipid. The cationic lipid may comprise any one or more cationic lipids disclosed herein. In a specific embodiment, the cationic lipid comprises ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). In some embodiments, the cationic lipid (e.g., ALC-0315) is present in an amount of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81 ．． 43,0.44,0.45,0.46,0.47,0.48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67, 0.68 , 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.9 3, 0.9 4.0.95 9, 1. 2,1.21,1.22,1.23,1.24,1.25,1.26,1.27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1.43,1.44,1 ．45,1 ．． 46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1 ．71、 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.9 6, 1.9 7, 1.98, 1.99, or 2 ng/μg/mg, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2 , 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0. 45, 0. 46,0.47,0.48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.7,0 ．71,0 ．． 72,0.73,0.74,0.75,0.76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96, 0.97 , 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.4 8, 1.4 9,1.5,1.51,1.52,1.53,1.54,1.55,1.56,1.57,1.58,1.59,1.6,1.61,1.62,1.63,1.64,1.65,1.66,1.67,1.68,1.69,1.7,1.71,1.72,1.73,1. 74, 1. 75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2n g/μg/mg, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 , 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0. 49, 0. 5.0.51 ．75,0 ．． 76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.0 1, 1. 02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.21,1.22,1.23,1.24,1.25,1.26, 1.27 , 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.5 2, 1.5 Between any two of the following: 3, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg , or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.4 9, 0.5 , 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0. 75, 0. 76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99,1,1.0 1, 1.0 2.1.03 ．27,1 ．． 28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1 ．53、 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg. In some embodiments, the cationic lipid (e.g., ALC-0315) has a molecular weight of at least 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.7 9, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL, up to 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0. 64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.4 9.0.5 74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0
0.97, 0.98, 0.99, or 1 mg/mL, or between any two of these, or exactly 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.70, 0.72, 0.74, 0.76, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL. 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, or 0.9-1. In some embodiments, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.4-0.45, 0.45-0.5, 0.5-0.55, 0.55-0.6, 0.6-0.65, 0.65-0.7, 0.7-0.75, 0.75-0.8, 0.8-0.85, 0.85-0.9, 0.9-0.95, or 0.95-1 mg/mL.

In a specific embodiment, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8-0.95 mg/mL. In a specific embodiment, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.8-0.9 mg/mL. In a specific embodiment, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.85-0.9 mg/mL. In a specific embodiment, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL. In a specific embodiment, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.86 mg/mL. The concentration for the lyophilized composition is determined after reconstitution.

In some embodiments, the VZV RNA-LNP compositions further comprise a PEGylated lipid (i.e., a PEG lipid). The PEGylated lipid may comprise any one or more of the PEGylated lipids disclosed herein. In a specific embodiment, the PEGylated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the PEGylated lipid (e.g., ALC-0159) is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42 , 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0. 67, 0. 68,0.69,0.7,0.71,0.72,0.73,0.74,0.75,0.76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0 ．93,0 ．． 94,0.95,0.96,0.97,0.98,0.99,1,1.01,1.02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1. 19, 1 ．． 2,1.21,1.22,1.23,1.24,1.25,1.26,1.27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1.43,1.44,1 ．４５、 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7 , 1.71 , 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1. 96, 1. 97, 1.98, 1.99, or 2 ng/μg/mg, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0. 2.0.21 ．45,0 ．． 46,0.47,0.48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.7,0 ．71、 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.9 6, 0.9 7.0.98 , 1.23 , 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1. 48, 1. 49,1.5,1.51,1.52,1.53,1.54,1.55,1.56,1.57,1.58,1.59,1.6,1.61,1.62,1.63,1.64,1.65,1.66,1.67,1.68,1.69,1.7,1.71,1.72,1.73,1 ．74,1 .75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23 ,0.2 4.0.25 ．49,0 ．． 5.0.51 ．75、 0.76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99,1,1 ．01,1 ．． 02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.21,1.22,1.23,1.24,1.25,1.26, 1.27 , 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.5 2, 1.5 Between any two of the following: 3, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg , or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.4 9, 0.5 , 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0. 75, 0. 76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99,1,1.0 1, 1.0 2.1.03 ．27,1 ．． 28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1 ．53、 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg. In some embodiments, the PEGylated lipid (e.g., ALC-0159) has a molecular weight of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 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, .39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33 , 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28 , 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL, or between any two of these, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0
0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some embodiments, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.4 mg/mL, at least 0.45 mg/mL, or at least 0.5 mg/mL. In some embodiments, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of 0.01-0.05, 0.05-0.1, 0.1-0.15, 0.15-0.2, or 0.2-0.25 mg/mL.

In a particular embodiment, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.05-0.15 mg/mL. In a particular embodiment, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.10-0.15 mg/mL. In a particular embodiment, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL. In a particular embodiment, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.11 mg/mL. Concentrations for lyophilized compositions are determined after reconstitution.

In some embodiments, the VZV RNA-LNP composition further comprises one or more structured lipids. The one or more structured lipids may comprise any one or more structured lipids disclosed herein. In a specific embodiment, the one or more structured lipids comprise a neutral lipid and a steroid or steroid analog. In a specific embodiment, the one or more structured lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. In some embodiments, the one or more structured lipids (e.g., DSPC and cholesterol) are at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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 ．． 41,0.42,0.43,0.44,0.45,0.46,0.47,0.48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65, 0.66 , 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.9 1, 0.9 2.0.93 7, 1.1 8,1.19,1.2,1.21,1.22,1.23,1.24,1.25,1.26,1.27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1. 43, 1. 44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7,1.71,1.72,1.73,1.74,1.75,1.76,1.77,1.78,1.79,1.8,1.81,1.82,1.83,1.84,1.85,1.86,1.87,1.88,1.89,1.9,1.91,1.92,1.93,1.94 , 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0 ．． 19,0.2,0.21,0.22,0.23,0.24,0.25,0.26,0.27,0.28,0.29,0.3,0.31,0.32,0.33,0.34,0.35,0.36,0.37,0.38,0.39,0.4,0.41,0.42,0.43,0 ．４４、 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.6 9, 0.7 , 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0. 95, 0. 96,0.97,0.98,0.99,1,1.01,1.02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.2 1, 1.2 2,1.23,1.24,1.25,1.26,1.27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1.43,1.44,1.45,1.46,1 ．４７．１ ．． 48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1 ．73、 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.9 8, 1.99 , or 2ng/μg/mg, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23 , 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0. 48, 0. 49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.7,0.71,0.72,0.73,0 ．74,0 ．． 75,0.76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99, 1, 1. 01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51 , 1.52 , 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg Between or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.2 4.0.25 ．49,0 ．． 5.0.51 ．75、 0.76,0.77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99,1,1 ．01, 1. 02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.21,1.22,1.23,1.24,1.25,1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52 , 1.53 , 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg. In some embodiments, the one or more structured lipids (e.g., DSPC and cholesterol) are at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, .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 mg/mL, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0. 27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, Between any two of the following: 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 mg/mL, or exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 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, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 6
0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some embodiments, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some embodiments, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of 0.05-0.1, 0.1-0.15, 0.15-0.2, 0.2-0.25, 0.25-0.3, 0.3-0.35, 0.35-0.4, 0.4-0.45, 0.45-0.5, 0.5-0.55, 0.55-0.6, 0.6-0.65, 0.65-0.7, 0.7-0.75, 0.75-0.8, 0.8-0.85, 0.85-0.9, 0.9-0.95, or 0.95-1 mg/mL.

In a specific embodiment, the one or more structured lipids include DSPC, and the DSPC is included in the composition at a concentration of about 0.1-0.25 mg/mL. In a specific embodiment, the one or more structured lipids include DSPC, and the DSPC is included in the composition at a concentration of about 0.15-0.25 mg/mL. In a specific embodiment, the one or more structured lipids include DSPC, and the DSPC is included in the composition at a concentration of about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL. In a specific embodiment, the DSPC is included in the composition at a concentration of about 0.19 mg/mL.

In a specific embodiment, the one or more structured lipids include cholesterol and the cholesterol is included in the composition at a concentration of about 0.3-0.45 mg/mL. In a specific embodiment, the one or more structured lipids include cholesterol and the cholesterol is included in the composition at a concentration of about 0.3-0.4. In a specific embodiment, the one or more structured lipids include cholesterol and the cholesterol is included in the composition at a concentration of about 0.35-0.45. In a specific embodiment, the one or more structured lipids include cholesterol and the cholesterol is included in the composition at a concentration of about 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 a specific embodiment, the cholesterol is included in the composition at a concentration of about 0.37 mg/mL. The concentration for the lyophilized composition is determined after reconstitution.

In some embodiments, the VZV RNA-LNP composition further comprises one or more buffering agents and stabilizers, and optionally a salt diluent. Thus, in some embodiments, the VZV RNA-LNP composition comprises a cationic lipid, a PEGylated lipid, one or more structured lipids, one or more buffering agents, stabilizers, and optionally a salt diluent.

In some embodiments, the VZV RNA-LNP composition comprises one or more buffering agents. The one or more buffering agents may comprise any one or more buffering agents disclosed herein. In specific embodiments, the composition comprises a Tris buffer comprising at least a first buffering agent and a second buffering agent. In some embodiments, the first buffering agent is tromethamine. In some embodiments, the second buffering agent is Tris hydrochloride (HCl). In some embodiments, the first buffering agent and the second buffering agent of the Tris buffer (e.g., tromethamine and Tris HCl) at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, ．． 48,0.49,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.7,0.71,0.72,0 ．73 , 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0. 98, 0 ．． 99,1,1.01,1.02,1.03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.21,1.22,1.23,1.2 4, 1 ．． 25,1.26,1.27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1.43,1.44,1.45,1.46,1.47,1.48,1.49, 1.5 , 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1. 75, 1 .76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μ g/mg, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24 , 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0. 49,0 ．． 5.0.51 ．75 , 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01 , 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1. 26, 1 ．． 27,1.28,1.29,1.3,1.31,1.32,1.33,1.34,1.35,1.36,1.37,1.38,1.39,1.4,1.41,1.42,1.43,1.44,1.45,1.46,1.47,1.48,1.49,1.5,1.51,1 ．52 , 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1. 77, 1 .78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.2 6, 0. 27.0.28 ．52、 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.7 7, 0. 78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.0 3, 1. 04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29 , 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.5 4,1. between or exactly any two of the following: 55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.2 5, 0. 26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0 ．51、 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.7 6, 0. 77,0.78,0.79,0.8,0.81,0.82,0.83,0.84,0.85,0.86,0.87,0.88,0.89,0.9,0.91,0.92,0.93,0.94,0.95,0.96,0.97,0.98,0.99,1,1.01,1.0 2, 1. 03,1.04,1.05,1.06,1.07,1.08,1.09,1.1,1.11,1.12,1.13,1.14,1.15,1.16,1.17,1.18,1.19,1.2,1.21,1.22,1.23,1.24,1.25,1.26,1.27, 1.28 , 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.5 3, 1. 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/μg/mg. The concentration of the lyophilized composition is determined after reconstitution.

In some embodiments, the VZV RNA-LNP composition is a liquid composition comprising a Tris buffer. In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is tromethamine. In some embodiments, the first buffer (e.g., tromethamine) is at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0. 21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48 , 0.49, or 0.5 mg/mL, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0 .24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/kg mL, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27 , 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL; or Exactly 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27 , 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some embodiments, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of at least 0.1, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, or at least 1 mg/mL. In some embodiments, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of 0.05-0.15, 0.15-0.25, 0.25-0.35, 0.35-0.45, 0.45-0.55, 0.55-0.65, 0.65-0.75, 0.75-0.85, or 0.85-0.95. In some embodiments, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of 0.05-0.1, 0.1-0.15, 0.15-0.2, 0.2-0.25, 0.25-0.3, 0.3-0.35, 0.35-0.4, 0.4-0.45, 0.45-0.5, 0.5-0.55, 0.55-0.6, 0.6-0.65, 0.65-0.7, 0.7-0.75, 0.75-0.8, 0.8-0.85, 0.85-0.9, 0.9-0.95, or 0.95-1 mg/mL.

In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of about 0.1-0.3 mg/mL. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of about 0.15-0.25 mg/mL. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3 mg/mL. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the liquid composition at a concentration of about 0.20 mg/mL.

In some embodiments, the VZV RNA-LNP composition is a liquid composition comprising a Tris buffer including a second buffer. In some embodiments, the second buffer comprises Tris HCl. In some embodiments, the second buffer (e.g., Tris HCl) is at least 0.5, 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1. 24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, or 1.5 mg/mL; Maximum 0.5, 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1 .26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, or 1.5 mg/mL, 0.5, 0.55, 1.1.01 .25, 1.26, 1.27, 1.2 Between any two of, or exactly 0.5, 8, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, or 1.5 mg/mL , 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1. 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, or 1.5 mg/mL. In some embodiments, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of at least 0.5, at least 0.55, at least 0.6, at least 0.65, at least 0.7, at least 0.75, at least 0.8, at least 0.85, at least 0.9, at least 0.95, at least 1, at least 1.05, at least 1.10, at least 1.15, at least 1.20, at least 1.25, at least 1.30, at least 1.35, at least 1.40, at least 1.45, or at least 1.50 mg/mL. In some embodiments, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, 0.9-1, 1-1.10, 1.10-1.20, 1.20-1.30, 1.30-1.40, or 1.40-1.50 mg/mL.

In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the liquid composition at a concentration of about 1.25-1.40 mg/mL. In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the liquid composition at a concentration of about 1.30-1.40 mg/mL. In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the liquid composition at a concentration of about 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, or 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL. In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the liquid composition at a concentration of about 1.32 mg/mL.

In some embodiments, the VZV RNA-LNP composition is a lyophilized composition comprising a Tris buffer. In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is tromethamine. In some embodiments, the first buffer (e.g., tromethamine) has a solubility of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 2.0.21 .45, 0.46, 0.47, 0. 48, 0.49, or 0.5 mg/mL, up to 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/ mL, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, Between any two of, or exactly between, 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 mg/mL 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.2 In some embodiments, the first buffering agent (e.g., tromethamine) is present in the lyophilized composition at a concentration of at least 0.8, 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 mg/mL after reconstitution. In some embodiments, the first buffering agent (e.g., tromethamine) is present in the lyophilized composition at a concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, or at least 0.5 mg/mL after reconstitution. In some embodiments, the first buffer (e.g., tromethamine (Tris base)) is included in the lyophilized composition at a concentration of 0.01-0.05, 0.05-0.1, 0.1-0.15, 0.15-0.2, 0.2-0.25 mg/mL, 0.25-0.3 mg/mL, 0.3-0.35 mg/mL, 0.35-0.4 mg/mL, 0.4-0.45 mg/mL, or 0.45-0.5 mg/mL after reconstitution.

In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the lyophilized composition at a concentration of about 0.01-0.15 mg/mL after reconstitution. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the lyophilized composition at a concentration of about 0.01-0.10 mg/mL after reconstitution. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the lyophilized composition at a concentration of about 0.05-0.15 mg/mL after reconstitution. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the lyophilized composition at a concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL after reconstitution. In a specific embodiment, the first buffering agent (e.g., tromethamine) is included in the lyophilized composition at a concentration of about 0.09 mg/mL after reconstitution.

In some embodiments, the VZV RNA-LNP composition is a lyophilized composition comprising a Tris buffer including a second buffer. In some embodiments, the second buffer comprises Tris HCl. In some embodiments, the second buffer (e.g., Tris HCl) has a pH of at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0. 7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/m L,max,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.7,0.71,0.72 , 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL, 0.1, 0.15, 0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0 .69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.7 Between any two of the following, or exactly 0.1, 0.5, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL ．． 15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.6,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.6 8, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, In some embodiments, the second buffer (e.g., Tris HCl) is present in the lyophilized composition at a concentration of at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 mg/mL after reconstitution. In some embodiments, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5, 0.5-0.6, 0.6-0.7, 0.7-0.8, 0.8-0.9, or 0.9-1 mg/mL after reconstitution.

In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the lyophilized composition at a concentration of about 0.5-0.65 mg/mL after reconstitution. In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the lyophilized composition at a concentration of about 0.5-0.6 mg/mL after reconstitution. In a specific embodiment, the second buffering agent (e.g., Tris HCl) is included in the lyophilized composition at a concentration of about 0.55-0.65 mg/mL after reconstitution. In a specific embodiment, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of about 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL after reconstitution. In a specific embodiment, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a concentration of about 0.57 mg/mL after reconstitution.

In some embodiments, the VZV RNA-LNP composition includes a stabilizing agent. The stabilizing agent may include any one or more stabilizing agents disclosed herein. In some embodiments, the stabilizing agent also functions as a cryoprotectant. In a specific embodiment, the stabilizing agent includes sucrose. In some embodiments, the stabilizing agent (e.g., sucrose) is at least 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 ... 1, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 10 8, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 , 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 15 3, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 , 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 , 199, or 200 ng/μg/mg, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 1 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96 , 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 1 12,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,14 2, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 1 57, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 ng/μ g/mg, 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, 6 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 , 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 ng/μg/mg, or or exactly 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, 6 2, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,1 47, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, It is included in the composition at a concentration of 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 ng/μg/mg.

In some embodiments, the VZV RNA-LNP composition is a liquid composition and the stabilizing agent (e.g., sucrose) is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 , 127, 128, 129 or 130 mg/mL, up to 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 mg/mL 0 mg/mL, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 mg/mL, or between any two of these or exactly 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129 or 130 mg/mL. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, or at least 130 mg/mL. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of 70-80, 80-90, 90-100, 100-110, 110-120, or 120-130 mg/mL.

In a particular embodiment, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of about 95-110 mg/mL. In a particular embodiment, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of about 95-105 mg/mL. In a particular embodiment, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of about 100-110 mg/mL. In a particular embodiment, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of about 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg/mL. In a particular embodiment, the stabilizing agent (e.g., sucrose) is included in the liquid composition at a concentration of about 103 mg/mL.

In some embodiments, the VZV RNA-LNP composition is a lyophilized composition, and the stabilizer (e.g., sucrose) is at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 102, 104, 105, 106, 107, 108, 109, 109, 109, 102, 103, 104, 105, 106, 107, 108, 109, 109, 1 3, 74, 75, 76, 77, 78, 79, or 80 mg/mL, up to 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or or 80 mg/mL, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 mg/mL, and between any two of these is included in the lyophilized composition at a concentration of exactly 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 mg/mL. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the lyophilized composition at a concentration of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 mg/mL after reconstitution. In some embodiments, the stabilizing agent (e.g., sucrose) is included in the lyophilized composition at a concentration of 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 mg/mL after reconstitution.

In a specific embodiment, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of about 35-50 mg/mL after reconstitution. In a specific embodiment, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of about 35-45 mg/mL after reconstitution. In a specific embodiment, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of about 40-50 mg/mL after reconstitution. In a specific embodiment, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL after reconstitution. In a specific embodiment, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a concentration of about 44 mg/mL after reconstitution.

In some embodiments, the lyophilized composition is reconstituted in a suitable carrier or diluent. The carrier or diluent may include any one or more of the carriers or diluents disclosed herein. In specific embodiments, the carrier or diluent includes a salt diluent, such as sodium chloride (NaCl) (e.g., saline, e.g., physiological or normal saline). The sodium chloride may include 0.9% sodium chloride for injection. In some embodiments, the lyophilized composition comprises at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0 .72,0. 73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.9 7, 0.98 , 0.99, or 1 mL, up to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0 ．49,0. 50,0.51,0.52,0.53,0.54,0.55,0.56,0.57,0.58,0.59,0.60,0.61,0.62,0.63,0.64,0.65,0.66,0.67,0.68,0.69,0.70,0.71,0.72,0.73,0.7 4, 0.75 , 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mL , 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26 , 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0 ．52, 0. 53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mL, and are exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0. 27.0.28 1, 0.52 , 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mL of saline solution. In some embodiments, the lyophilized composition is reconstituted in at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 mL of sodium chloride.

In a specific embodiment, the lyophilized composition is reconstituted in about 0.6-0.75 mL of sodium chloride/saline. In a specific embodiment, the lyophilized composition is reconstituted in about 0.65-0.75 mL of sodium chloride/saline. In a specific embodiment, the lyophilized composition is reconstituted in about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74 or 0.75 mL of sodium chloride/saline.

In some embodiments, the salt diluent (e.g., NaCl) is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23 , 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50 ng/μg/mg, up to 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50 ng/μg /mg, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, Between or exactly any two of the following: 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50 ng/μg/mg 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50 ng/μg/mg. In some embodiments, the salt diluent (e.g., NaCl) has at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19 , 19.5, or 20 mg/mL, up to 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/mL , 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/mL In some embodiments, the salt diluent (e.g., NaCl) is present in the lyophilized composition at a concentration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 mg/mL after reconstitution.

In a specific embodiment, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a concentration of about 5-15 mg/mL after reconstitution. In some embodiments, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a concentration of about 5-10 mg/mL after reconstitution. In a specific embodiment, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL after reconstitution. In a specific embodiment, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a concentration of about 9 mg/mL after reconstitution.

The pH of the VZV RNA-LNP composition is at least pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, and up to ... 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein between any two of pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In some embodiments, the VZV RNA-LNP composition has a pH of at least 6.5, at least 7.0, at least 7.5, at least 8.0, or at least 8.5. In specific embodiments, the VZV RNA-LNP composition has a pH of 6.0-7.5, 6.5-7.5, 7.0-8.0, 7.5-8.5. In specific embodiments, the VZV RNA-LNP composition has a pH of 7.0-8.0. In specific embodiments, the VZV RNA-LNP composition has a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In specific embodiments, the VZV RNA-LNP composition has a pH of about 7.4. In some embodiments, sodium hydroxide buffer may be used for buffer pH adjustment.

In a specific embodiment, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein encapsulated in an LNP having a lipid composition of cationic lipid at a concentration of about 0.8-0.95 mg/mL, PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, first structural lipid at a concentration of about 0.1-0.25 mg/mL, and second structural lipid at a concentration of about 0.3-0.45 mg/mL.

In a specific embodiment, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein encapsulated in LNPs having a lipid composition of ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, and cholesterol at a concentration of about 0.3-0.45 mg/mL.

In a specific embodiment, the VZV RNA-LNP composition is a liquid VZV RNA-LNP composition, and the liquid VZV RNA-LNP composition further comprises a buffer composition comprising a first buffer at a concentration of about 0.15-0.3 mg/mL, a second buffer at a concentration of about 1.25-1.4 mg/mL, and a stabilizer at a concentration of about 95-110 mg/mL. In a specific embodiment, the VZV RNA-LNP composition is a liquid VZV RNA-LNP composition, and the liquid VZV RNA-LNP composition further comprises a Tris buffer composition comprising tromethamine at a concentration of about 0.1-0.3 mg/mL, Tris HCl at a concentration of about 1.25-1.4 mg/mL, and sucrose at a concentration of about 95-110 mg/mL.

Thus, in a specific embodiment, the liquid VZV RNA-LNP composition comprises a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a first structural lipid at a concentration of about 0.1-0.25 mg/mL, a second structural lipid at a concentration of about 0.3-0.45 mg/mL, and further comprises a first buffering agent at a concentration of about 0.1-0.3 mg/mL, a second buffering agent at a concentration of about 1.25-1.4 mg/mL, and a stabilizing agent at a concentration of about 95-110 mg/mL.

Thus, in a specific embodiment, the liquid VZV RNA-LNP composition comprises ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, cholesterol at a concentration of about 0.3-0.45 mg/mL, and further comprises a Tris buffer composition comprising tromethamine at a concentration of about 0.1-0.3 mg/mL, Tris HCl at a concentration of about 1.25-1.4 mg/mL, and sucrose at a concentration of about 95-110 mg/mL.

In a specific embodiment, the VZV RNA-LNP composition is a lyophilized VZV RNA-LNP composition, and the lyophilized VZV RNA-LNP composition (after reconstitution) further comprises a first buffering agent at a concentration of about 0.01-0.15 mg/mL, a second buffering agent at a concentration of about 0.5-0.65 mg/mL, a stabilizer at a concentration of about 35-50 mg/mL, and a salt diluent at a concentration of about 5-15 mg/mL.

In a specific embodiment, the VZV RNA-LNP composition is a lyophilized VZV RNA-LNP composition, and the lyophilized VZV RNA-LNP composition (after reconstitution) further comprises tromethamine at a concentration of about 0.01-0.15 mg/mL, a Tris buffer composition comprising Tris HCl at a concentration of about 0.5-0.65 mg/mL, sucrose at a concentration of about 35-50 mg/mL, and sodium chloride (NaCl) at a concentration of about 5-15 mg/mL.

Thus, in a specific embodiment, the lyophilized VZV RNA-LNP composition (after reconstitution) comprises a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a first structural lipid at a concentration of about 0.1-0.25 mg/mL, a second structural lipid at a concentration of about 0.3-0.45 mg/mL, and further comprises a first buffer at a concentration of about 0.01-0.15 mg/mL, a second buffer at a concentration of about 0.5-0.65 mg/mL, a stabilizer at a concentration of about 35-50 mg/mL, and a salt diluent at a concentration of about 5-15 mg/mL. In a specific embodiment, the lyophilized composition is reconstituted in 0.6-0.75 mL of salt diluent.

Thus, in some embodiments, the lyophilized VZV RNA-LNP composition (after reconstitution) comprises ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, cholesterol at a concentration of about 0.3-0.45 mg/mL, and further comprises tromethamine at a concentration of about 0.01-0.15 mg/mL, Tris HCl at a concentration of about 0.5-0.65 mg/mL, sucrose at a concentration of about 35-50 mg/mL, and NaCl at a concentration of about 5-15 mg/mL. In a specific embodiment, the lyophilized composition is reconstituted in 0.6-0.75 mL of NaCl (saline).

The concentration in the lyophilized VZV RNA-LNP composition is determined after reconstitution.

In some embodiments, the VZV RNA-LNP composition (prior to lyophilization) comprises a cationic lipid at a concentration of about 1.0-3.0 mg/mL, a PEGylated lipid at a concentration of about 0.10-0.35 mg/mL, a first structural lipid at a concentration of about 0.4-0.55 mg/mL, a second structural lipid at a concentration of about 0.85-1.0 mg/mL, and further comprises a first buffer at a concentration of about 0.1-0.3 mg/mL, a second buffer at a concentration of about 1.25-1.40 mg/mL, and a stabilizer at a concentration of about 95-110 mg/mL.

Thus, in some embodiments, the VZV RNA-LNP composition comprises (prior to lyophilization) ALC-0315 at a concentration of about 1.0-3.0 mg/mL, ALC-0159 at a concentration of about 0.10-0.35 mg/mL, DSPC at a concentration of about 0.4-0.55 mg/mL, cholesterol at a concentration of about 0.85-1.0 mg/mL, and further comprises tromethamine at a concentration of about 0.1-0.3 mg/mL, Tris HCl at a concentration of about 1.25-1.40 mg/mL, and sucrose at a concentration of about 95-110 mg/mL.

The VZV RNA-LNP composition further comprises VZV RNA as described herein encapsulated in LNPs. See Section D. Administration.

In a specific embodiment, the VZV RNA-LNP composition comprises a lipid composition of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, encapsulated in LNPs having a lipid composition of cationic lipid at a concentration of about 0.8-0.95 mg/mL, PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, first structural lipid at a concentration of about 0.1-0.25 mg/mL, and second structural lipid at a concentration of about 0.3-0.45 mg/mL. A liquid VZV RNA-LNP composition comprising a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein at a concentration of 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, and further comprising a buffer composition comprising a first buffer at a concentration of about 0.15-0.3 mg/mL, a second buffer at a concentration of about 1.25-1.4 mg/mL, and a stabilizer at a concentration of about 95-110 mg/mL.

In a specific embodiment, the liquid VZV RNA-LNP composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, and up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in LNPs having a lipid composition of ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, and cholesterol at a concentration of about 0.3-0.45 mg/mL. 0.01-0.09mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90mg/mL, preferably about 0.01-0.09mg/mL, more preferably about 0.06mg/mL, The composition comprises an RNA polynucleotide, and further comprises a Tris buffer composition comprising tromethamine at a concentration of about 0.1-0.3 mg/mL, Tris HCl at a concentration of about 1.25-1.4 mg/mL, and sucrose at a concentration of about 95-110 mg/mL.

In a specific embodiment, the VZV RNA-LNP composition comprises a lipid composition of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to 0.01, 0.25, 0.45, or 0.60 mg/mL, 0.5, or 0.80 mg/mL, 0.60 mg/mL, or 0.75 mg/mL, 0.85 mg/mL, or 0.95 mg/mL, 0.95 mg/mL, or 0.85 mg/mL, or 0.9 ... and a lyophilized VZV RNA-LNP composition comprising a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein at a concentration of 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, and further comprising a first buffering agent at a concentration of about 0.01-0.15 mg/mL, a second buffering agent at a concentration of about 0.5-0.65 mg/mL, a stabilizer at a concentration of about 35-50 mg/mL, and a salt diluent at a concentration of 5-15 mg/mL. In a specific embodiment, the lyophilized composition is reconstituted in 0.6-0.75 mL of salt diluent. The concentration in the lyophilized VZV RNA-LNP composition is determined after reconstitution.

In a specific embodiment, the lyophilized VZV RNA-LNP composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in LNPs having a lipid composition of ALC-0315 at a concentration of about 0.8-0.95 mg/mL, ALC-0159 at a concentration of about 0.05-0.15 mg/mL, DSPC at a concentration of about 0.1-0.25 mg/mL, and cholesterol at a concentration of about 0.3-0.45 mg/mL. 0.01-0.09 mg/mL, more preferably about 0.06 mg/mL, and further comprising tromethamine at a concentration of about 0.01-0.15 mg/mL, Tris HCl at a concentration of about 0.5-0.65 mg/mL, sucrose at a concentration of about 35-50 mg/mL, and NaCl at a concentration of about 5-15 mg/mL. In a specific embodiment, the lyophilized composition is reconstituted in 0.6-0.75 mL of NaCl diluent (saline). The concentration in the lyophilized VZV RNA-LNP composition is determined after reconstitution.

In some embodiments, the VZV RNA-LNP composition (prior to lyophilization) comprises a lipid composition of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.0, encapsulated in LNPs having a lipid composition of cationic lipid at a concentration of about 1.0-3.0 mg/mL, PEGylated lipid at a concentration of about 0.10-0.35 mg/mL, first structural lipid at a concentration of about 0.4-0.55 mg/mL, and second structural lipid at a concentration of about 0.85-1.0 mg/mL. The composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein at a concentration of 1, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, and further comprises a first buffering agent at a concentration of about 0.1-0.3 mg/mL, a second buffering agent at a concentration of about 1.25-1.40 mg/mL, and a stabilizing agent at a concentration of about 95-110 mg/mL.

Thus, in some embodiments, the VZV RNA-LNP composition (before lyophilization) comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, and up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, encapsulated in LNPs having a lipid composition of ALC-0315 at a concentration of about 1.0-3.0 mg/mL, ALC-0159 at a concentration of about 0.10-0.35 mg/mL, DSPC at a concentration of about 0.4-0.55 mg/mL, and cholesterol at a concentration of about 0.85-1.0 mg/mL. The composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide disclosed herein at a concentration of 0, 0.45, 0.60, 0.75, or 0.90 mg/mL, or exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01 to 0.09 mg/mL, more preferably 0.15 mg/mL, and further comprises tromethamine at a concentration of about 0.1 to 0.3 mg/mL, Tris HCl at a concentration of about 1.25 to 1.40 mg/mL, and sucrose at a concentration of about 95 to 110 mg/mL.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, of RNA encapsulated in LNPs; or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, of an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, and further comprising about 5-15 mM Tris buffer, 200-400 mM sucrose at a pH of about 7.0-8.0.

In some embodiments, the liquid RNA-LNP immunogenic composition comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL of RNA encapsulated in LNPs. The composition comprises an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein at a concentration between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01 to 0.09 mg/mL, more preferably about 0.06 mg/mL, and further comprises about 10 mM Tris buffer, 300 mM sucrose at a pH of about 7.4.

In some embodiments, the RNA-LNP immunogenic composition (before lyophilization) comprises at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or 0.01, 0.15, It comprises an RNA molecule/polynucleotide encoding a VZV polypeptide disclosed herein at a concentration between any two of 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, and further comprises about 5-15 mM Tris buffer, 200-400 mM sucrose at a pH of about 7.0-8.0, and is reconstituted with a 0.9% sodium chloride diluent.

In some embodiments, the RNA-LNP immunogenic composition (before lyophilization) has at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or 0.01, 0.15, 0.3 It comprises an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein at a concentration between any two of 0, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01-0.09 mg/mL, more preferably 0.15 mg/mL, and further comprises about 10 mM Tris buffer, 300 mM sucrose at a pH of about 7.4, and is reconstituted with a 0.9% sodium chloride diluent.

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

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

The present disclosure includes a method of preventing, treating, or ameliorating an infection, disease, or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule or composition comprising at least one open reading frame encoding a polypeptide as described herein. As such, the present disclosure contemplates vaccines for use in both active and passive immunization aspects. Immunogenic compositions proposed to be suitable for use as vaccines may be prepared from RNA molecules encoding polypeptides, such as VZV glycoproteins. In certain embodiments, the immunogenic compositions are lyophilized for more ready formulation into a desired medium.

The preparation of vaccines containing nucleic acids and/or peptides or polypeptides as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for dissolution or suspension in liquid prior to injection may also be prepared. The preparations may also be emulsified. The active immunogenic ingredient is often mixed with an excipient that is pharma- ceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, or ethanol, and combinations thereof. Additionally, if desired, the vaccine may contain certain amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccine. In a specific embodiment, the vaccine is formulated with a combination of substances as described in U.S. Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.

Vaccines may be conventionally administered parenterally, for example, by injection, either subcutaneously or intramuscularly. Additional formulations suitable for other modes of administration include suppositories, and in some cases oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides, and such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%. In some embodiments, suppositories may be formed from mixtures containing the active ingredient in the range of about 1% to about 2%. Oral formulations include commonly employed excipients such as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and contain about 10% to about 95% of the active ingredient.

The nucleic acid constructs encoding the polypeptides and the polypeptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the peptide) and those formed with inorganic acids, such as hydrochloric or phosphoric acids, or organic acids, such as acetic acid, oxalic acid, tartaric acid, and mandelic acid.

Typically, the vaccine is administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The amount administered will depend on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. The precise amount of active ingredient required to be administered will depend on the physician's judgment. However, suitable dosage ranges are on the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable, but an initial administration, followed by subsequent inoculations or other administrations are typical.

The mode of application may vary widely. Any of the conventional methods for administration of vaccines are applicable. These would include oral application in a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, and by injection. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.

In certain embodiments, it may be desirable to administer a single dose of the vaccine. In some embodiments, it may be desirable to administer multiple doses of the vaccine, e.g., 2, 3, 4, 5, 6 or more doses. Vaccinations may be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, including all ranges in between. In some embodiments, vaccinations may be administered at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, including all ranges in between. Periodic boosters at 1-5 year intervals may be desirable to maintain protective levels of antibodies.

i. Carrier The pharma-ceutically acceptable carrier may comprise liquid or non-liquid based compositions. When the composition is provided in liquid form, the carrier may be water, e.g. pyrogen-free water; isotonic saline or buffered (aqueous) solution, e.g. phosphate, citrate buffer solution. Water or buffers, e.g. aqueous buffers, containing sodium salts, calcium salts, and/or potassium salts may be used. Sodium, calcium and/or potassium salts may be present in the form of their halides, e.g. chloride, iodide or bromide, their hydroxides, carbonates, bicarbonates or sulfates, etc. Examples of sodium salts include, but are not limited to, NaCl, NaI, _NaBr , _Na2CO3 , _NaHCO3 , _Na2SO4 , _{Na2HPO4 , Na2HPO4.2H2O} , examples of potassium salts include, but are not limited to, KCl, KI, _KBr , _K2CO3 , _{KHCO3 , K2SO4} , _KH2PO4 , and examples of calcium salts include, but are not limited to, _CaCl2 , _{Cal2 , CaBr2 , CaCO3 , CaSO4 ,} Ca(OH _{) 2} . Examples of further carriers can include sugars such as lactose, glucose, trehalose and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; tallow; solid lubricants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cacao oil; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid. Examples of further carriers can include colloidal silicon oxide, magnesium stearate, cellulose and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents as well as pharmaceutical requirements for their use are described in Remington's Pharmaceutical Sciences.

ii. Adjuvants Suitable adjuvants include any acceptable immunostimulatory compound, such as cytokines, toxins, or synthetic compositions. Numerous adjuvants may be used to enhance antibody responses. Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include Freund's adjuvant, oils such as MONTANIDE® ISA51, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, alpha-interferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts such as aluminum hydroxide or other aluminum compounds, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), lipopeptides (e.g., Pam3Cys), RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. Even MHC antigens may be used.

Various methods of achieving an adjuvant effect for vaccines include the use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about a 0.05 to about a 0.1% solution in phosphate buffered saline, mixed with a synthetic polymer of sugar (CARBOPOL®), used as about a 0.25% solution, and aggregation of proteins in the vaccine by heat treatment at temperatures ranging from about 70°C to about 101°C for periods of 30 seconds to 2 minutes, respectively. Agglutination by reactivation with pepsin-treated (Fab) antibodies against albumin; mixtures with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of gram-negative bacteria; emulsions in physiologically acceptable oil vehicles (e.g., mannide monooleate (Aracel A)); or emulsions with a 20% solution of perfluorocarbon used as a block substitute (FLUOSOL-DA®) may also be used to produce an adjuvant effect.

In addition to adjuvants, it may be desirable to co-administer a biologic response modifier (BRM) to enhance the immune response. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose cyclophosphamide (CYP; 300 mg/m ² ) (Johnson/Mead, NJ) and genes encoding cytokines, such as gamma-interferon, IL-2, or IL-12, or proteins involved in immune helper function, such as B-7.

C. Combination therapy The compositions and related methods of the present disclosure, particularly the administration of RNA molecules encoding VZV polypeptides, may also be used in combination with the administration of traditional therapies.These include, but are not limited to, the administration of antiviral therapies, such as acyclovir, valacyclovir, and famciclovir, or various combinations of antiviral agents.Also include the administration of one or more therapies to treat one or more symptoms of VZV infection, including, but not limited to, steroids, including corticosteroids, anti-inflammatory agents, including acetaminophen or ibuprofen, pain relievers, creams or lotions to relieve itching, cold compresses, or various combinations thereof.

In one embodiment, it is contemplated that the vaccine and/or therapy is used in combination with an antiviral treatment. Alternatively, the therapy may precede or follow treatment with the other agent by intervals ranging from minutes to weeks. In embodiments in which the other agent and/or vaccine are administered separately, it is generally done to ensure that no significant period of time elapses between the time of delivery of each to ensure that the agent and the immunogenic composition can still exert their advantageously combined effect in the subject. In such embodiments, it is contemplated that both modalities may be administered within about 12-24 hours of each other or within about 6-12 hours of each other. In some circumstances, it may be desirable to significantly extend the period for administration such that several days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks) elapse between each administration.

Various combinations may be used, for example, immunogenic polypeptides provided as part of an antiviral therapy "A" and an immunotherapeutic regime "B":
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A
B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Administration of the immunogenic compositions of the present disclosure to patients/subjects will follow general protocols for administration of VZV RNA vaccine compositions, or other compositions described herein, taking into account any toxicities of such compounds. It is expected that treatment cycles will be repeated as necessary. It is also envisioned that various standard therapies, such as hydration, may be applied in combination with the therapies described.

D. Administration Administration of the compositions described herein may be performed via any of the accepted modes of administration of agents serving similar purposes. In some embodiments, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In a specific embodiment, the VZV RNA molecules and/or RNA-LNP compositions are administered intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration with absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than through the gastrointestinal tract, for example, by intravenous injection. In one embodiment, the pharmaceutical composition is formulated for intramuscular administration. In another embodiment, the pharmaceutical composition is formulated for systemic administration, for example, intravenous administration.

Pharmaceutical compositions may be formulated into solid, semi-solid, liquid, lyophilized, frozen, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intradermal, intrasternal injection, or infusion techniques. The pharmaceutical compositions described herein are formulated to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. The composition administered to a subject or patient takes the form of one or more dosage units, for example, a tablet may be a single dosage unit, and a container of the compound in aerosol form may hold multiple dosage units. The composition to be administered will, in each case, contain a therapeutically and/or prophylactically effective amount of a compound within the scope of this disclosure, or a pharma- ceutically acceptable salt thereof, for treatment of the disease or condition of interest according to the teachings set forth herein.

Pharmaceutical compositions within the scope of the present disclosure may be in solid or liquid form and may be frozen or lyophilized. In one embodiment, the carrier is particulate, so that the composition is, for example, in tablet or powder form. The carrier may be liquid, so that the composition may be, for example, an oral syrup, an injectable liquid, or an aerosol, useful, for example, in inhalation administration. In some embodiments, when intended for oral administration, the pharmaceutical composition is in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included in the forms considered herein as either solid or liquid. As a solid composition for oral administration, the pharmaceutical composition may be formulated into forms such as powders, granules, compressed tablets, pills, capsules, chewing gum, or wafers. Such solid compositions typically contain one or more inert diluents or edible carriers. Additionally, one or more of the following may be present or excluded: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, PRIMOJEL®, and corn starch; lubricants, such as magnesium stearate or STEROTEX®; lubricants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate, or orange flavoring; and colorings. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it may contain, in addition to the above types of materials, a liquid carrier, such as polyethylene glycol or oil. The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion, or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. In some embodiments, when intended for oral administration, the composition contains, in addition to the compound of the present disclosure, one or more of a sweetener, a preservative, a dye/colorant, and a flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer, and an isotonic agent may be included or excluded.

Liquid pharmaceutical compositions, whether in solution, suspension, or other similar form, may contain or exclude one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions, such as saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that may serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose; agents to act as cryoprotectants, such as sucrose or trehalose. Parenteral preparations may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In one embodiment, saline is the adjuvant. In one embodiment, the injectable pharmaceutical composition is sterile. A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of the compound such that a suitable dosage will be obtained.

Pharmaceutical compositions may be prepared by methodologies well known in the pharmaceutical arts. For example, pharmaceutical compositions intended to be administered by injection may be prepared by combining a nucleic acid or polypeptide with sterile distilled water or other carrier to form a solution. A surfactant may be added to facilitate the formation of a homogenous solution or suspension. Surfactants are compounds that non-covalently interact with compounds consistent with the teachings herein to facilitate dissolution or homogenous suspension of the compound in an aqueous delivery system.

The pharmaceutical compositions according to the present disclosure, or pharma- ceutically acceptable salts thereof, are generally applied in a "therapeutically effective amount" or a "prophylactically effective amount" and in a "pharmaceutically acceptable preparation". The term "pharmaceutically acceptable" refers to the non-toxicity of the material that does not interact with the action of the active components of the pharmaceutical composition. The terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that achieves the desired reaction or the desired effect alone or together with further doses. In the case of the treatment of a particular disease, in one embodiment, the desired reaction relates to the inhibition of the course of the disease. This includes the slowing down of the progression of the disease, and in particular, the prevention or reversal of the progression of the disease. The desired reaction in the treatment of a disease may also be the delay of the onset or the prevention of the onset of said disease or said condition.

Compositions within the scope of the present disclosure are administered in therapeutically and/or prophylactically effective amounts, which will vary depending on a variety of factors, including the activity of the particular therapeutic and/or prophylactic agent used; the metabolic stability and length of action of the therapeutic and/or prophylactic agent; the patient's individual parameters, including age, weight, general health, sex, and diet; the mode, time, and/or duration of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. Thus, the administered dose of the compositions described herein may depend on a variety of such parameters. If the response in the patient is inadequate at the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used. In some embodiments, the composition (e.g., VZV The RNA-LNP composition) may be administered at a dose of from 0.0001 ng/μg/mg per kg to 100 ng/μg/mg per kg, from 0.001 ng/μg/mg per kg to 0.05 ng/μg/mg per kg, from 0.005 ng/μg/mg per kg to 0.05 ng/μg/mg per kg, from 0.001 ng/μg/mg per kg to 0.005 ng/μg/mg per kg, from 0.05 ng/μg/mg per kg to 0.5 ng/μg/mg per kg, from 0. ...5 ng/μg/mg per kg to 0.5 ng/μg/mg per kg, from 0.05 ng/μg/mg per kg to 0.5 ng/μg/mg per kg, from 0.05 ng/μg/mg per kg to 0.5 ng/μg/mg per kg, In some embodiments, the composition (e.g., VZV) may be administered at a dosage level sufficient to deliver a dose of from 0.1 ng/μg/mg per kg to 50 ng/μg/mg per kg, from 0.1 ng/μg/mg per kg to 40 ng/μg/mg per kg, from 0.5 ng/μg/mg per kg to 30 ng/μg/mg per kg, from 0.01 ng/μg/mg per kg to 10 ng/μg/mg per kg, from 0.1 ng/μg/mg per kg to 10 ng/μg/mg per kg, or from 1 ng/μg/mg per kg to 25 ng/μg/mg per kg (see, e.g., the unit dose ranges set forth in WO 2013/078199, which is incorporated herein by reference in its entirety). The RNA-LNP composition) is administered at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 3 9, 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, 8 4, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 6, 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 ng/μg/mg, exactly 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.00 7, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 1 7, 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 ng/μg/mg, or 0.0 001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0. 02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg.

In some embodiments, the composition (e.g., a VZV RNA-LNP composition) is administered at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 250, 300, 450, 500, 600, 700, 800, 900, 1000, 1500, 2500, 3000, 4500, 50 ... 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 4 3, 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 ng/μg/mg, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003 , 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 4, 95, 96, 97, 98, 99, or 100 ng/μg/mg, exactly 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0. 009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 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 ng/μg/mg, or 0.0001, 0.000 2, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0 .04, 0.05, 0.06 , 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 3 It may be administered at a total dose or dosage level sufficient to deliver a total dose between any two of 1, 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 ng/μg/mg.

In specific embodiments, the composition (e.g., the VZV RNA-LNP composition) has at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.00 8, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15,16, 17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 7, 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 m g/mL, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0. 01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 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 mg/mL, exactly 0. 0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0 ．02, 0. 03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 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, 6 6, 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 mg/mL, or 0.0001, 0.000 2, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0 .04, 0.0 5, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2 7, 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 mg/mL of VZV encapsulated in LNPs. It may be administered at a total dose or dosage level sufficient to deliver the total dose of RNA.

In exemplary aspects, a composition (e.g., a VZV RNA-LNP composition) may be administered at a dose level of VZV RNA encapsulated in the LNP of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL. In exemplary embodiments, the composition (e.g., a VZV RNA-LNP composition) may be administered at a dose level of at least 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg, up to 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg, or between any two of 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg of VZV RNA encapsulated in the LNP.

In specific embodiments, the composition (e.g., the VZV RNA-LNP composition) has at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.00 8, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 ,15,16, 17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 7, 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 μ g/mL, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0. 01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 ,61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg/mL, exactly 0. 0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0 ．02, 0. 03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 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, 6 6, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg/mL, or 0.0001, 0.000 2, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0 .04, 0.0 5, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2 7, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μg/mL of VZV encapsulated in LNPs. It may be administered at a total dose or dosage level sufficient to deliver the total dose of RNA.

In exemplary aspects, a composition (e.g., a VZV RNA-LNP composition) may be administered at a dosage level of at least 1, 15, 30, 45, 60, 75, 90, 100, up to 1, 15, 30, 45, 60, 75, 90, 100, exactly 1, 15, 30, 45, 60, 75, 90, 100, or between any two of 1, 15, 30, 45, 60, 75, 90, 100 or higher μg/mL of VZV RNA encapsulated in the LNP. In exemplary embodiments, the composition (e.g., a VZV RNA-LNP composition) may be administered at a dose level of at least 1, 15, 30, 45, 60, 75, 90, 100, up to 1, 15, 30, 45, 60, 75, 90, 100, exactly 1, 15, 30, 45, 60, 75, 90, 100, or between any two of 1, 15, 30, 45, 60, 75, 90, 100 or higher μg of VZV RNA encapsulated in the LNP.

The desired dosage may be delivered multiple times per day (e.g., 1, 2, 3, 4, 5, or more times per day), every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In certain embodiments, the desired dosage may be delivered using a single dose administration. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more administrations). When multiple administrations are used, a split dosing regimen may be used. The time between the initial administration of the composition and the subsequent administration of the composition can be 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week , 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years, but is not limited thereto.

In some embodiments, the composition (e.g., a VZV RNA-LNP composition) may be administered in a single dose. In some embodiments, the composition (e.g., a VZV RNA-LNP composition) may be administered twice (e.g., on days 0 and about 7, 0 and about 14, 0 and about 21, 0 and about 28, 0 and about 60, 0 and about 90, 0 and about 120, 0 and about 150, 0 and about 180, 0 and about 1 month later, 0 and about 2 months later, 0 and about 3 months later, 0 and about 6 months later, 0 and about 9 months later, 0 and about 12 months later, 0 and about 18 months later, 0 and about 2 years later, 0 and about 5 years later, or 0 and about 10 years later), each administration being at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0008, 0.0009, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0001, 0.0002, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0001, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0 ．． 0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0. 09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,3 1, 32, 33, 34, 35, 36, 37, 38, 3 9, 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, 8 7, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0. 005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg, to the exact value of 0.0001, 0.0002, 0.00 03, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0. 05, 0.06, 0.07, 0.08, 0.09, 0 ．． 1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32, 33, 34, 35, 36, 37, 38, 39, 4 0, 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 ng/μg/mg, or 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005 , 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,5 7, 58, 59, 60, 61, 62, 63, 64, 6 A total dose or dosage level sufficient to deliver a total dose of VZV RNA encapsulated in the LNPs between any two of 5, 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 ng/μg/mg may be used. Higher and lower dosages and frequencies of administration are encompassed by the present disclosure. For example, the composition (e.g., the VZV RNA-LNP composition) may be administered three or four times. Periodic boosters at 1-5 year intervals may be desired to maintain protective levels of antibodies.

In some embodiments, a composition (e.g., a VZV RNA-LNP composition) has at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008 , 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 0, 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 ng/μ g/mg, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.0 1,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,1 8, 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, 6 1, 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 ng/μg/mg, or exactly 0. 0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0 .02, 0.0 3,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 , 22, 23, 2 4, 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 ng/μg/mg, or 0.0001, 0.000 2, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0 .04, 0.0 5,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg of VZV encapsulated in LNPs. In some embodiments, the composition (e.g., the VZV RNA-LNP composition) is administered to the subject as a single dose of VZV RNA encapsulated in LNPs of at least 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, up to 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, exactly 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or between any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more.

In some embodiments, a composition (e.g., a VZV RNA-LNP composition) has at least 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008 , 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 0, 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 ng/μ g/mg, up to 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.0 1,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,1 8, 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, 6 1, 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 ng/μg/mg, or exactly 0. 0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0 .02, 0.0 3,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21 , 22, 23, 2 4, 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 ng/μg/mg, or 0.0001, 0.000 2, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0 .04, 0.0 5, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/μg/mg of VZV encapsulated in LNPs. In some embodiments, the composition (e.g., the VZV RNA-LNP composition) is administered to the subject as two doses of VZV RNA encapsulated in LNPs of at least 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, up to 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, exactly 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or between any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more.

In a specific embodiment, the composition (e.g., VZV The RNA-LNP composition) may be administered twice (e.g., on days 0 and 28, on days 0 and 60, on days 0 and 180, on days 0 and 2 months later, on days 0 and 6 months later), with each administration providing at least 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, up to 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg, or between or more of any two of 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg. This may be done at a total dose or dosage level sufficient to deliver the total dose of RNA.

IX. METHODS OF USE Provided herein are compositions (e.g., pharmaceutical compositions comprising VZV RNA molecules and/or VZV RNA-LNPs), methods, kits, and reagents for the prevention and/or treatment of VZV in humans and other mammals.

VZV RNA compositions (e.g., VZV RNA-LNP compositions) may be used as therapeutic or prophylactic agents. They may be used in medicines to prevent and/or treat infectious diseases. In an exemplary embodiment, the VZV RNA compositions are used to provide prophylactic protection from chickenpox or herpes zoster. Chickenpox is an acute infectious disease caused by VZV. The VZV vaccines of the present disclosure may be used to prevent and/or treat VZV (shingles or herpes zoster) and may be particularly useful for the prevention and/or treatment of immunocompromised and elderly patients to prevent or reduce the severity and/or duration of herpes zoster.

In some embodiments, a VZV RNA composition (e.g., a VZV RNA-LNP composition) of the present disclosure is administered to a subject (e.g., a mammalian subject, e.g., a human subject), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide.

In some embodiments, the VZV RNA compositions of the present disclosure may be used to prime immune effector cells, for example, by activating peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (or re-infused) into a subject.

In some embodiments, following administration of a VZV RNA molecule described herein, for example formulated as an RNA-LNP, at least a portion of the RNA is delivered to a target cell. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce its encoded polypeptide or protein. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell, for example a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell or a macrophage. The RNA molecules described herein, for example RNA-LNPs, may be used to deliver RNA to such target cells. Thus, the present disclosure also relates to a method of delivering RNA to a target cell in a subject, comprising administering to the subject an RNA-particle described herein.

In some embodiments, the RNA is delivered to the cytosol of the target cell. In some embodiments, the RNA is translated by the target cell to produce a polypeptide or protein encoded by the RNA. "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and having resulting biological properties. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, may be referred to as encoding the protein or other product of that gene or cDNA.

In some embodiments, the nucleic acid compositions described herein, e.g., compositions comprising VZV RNA-LNPs, are characterized by sustained expression of the encoded polypeptide (e.g., when administered to a subject). For example, in some embodiments, such compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from such a human, and in some embodiments, such expression persists for a period of at least 36 hours or longer, including, e.g., at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

In some aspects, the present disclosure relates to a method of inducing an immune response to VZV in a subject. The method includes administering to the subject an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another aspect, the present disclosure relates to a method of vaccinating a subject, the method comprising administering to a subject in need thereof an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another aspect, the present disclosure relates to a method of treating or preventing an infectious disease, the method comprising administering to a subject an effective amount of an RNA molecule, RNA-LNP and/or composition described herein.

In another aspect, the disclosure relates to a method of treating, preventing, or reducing the severity of a VZV infection and/or disease caused by VZV. The method comprises administering to a subject an effective amount of an RNA molecule, RNA-LNP, and/or composition described herein.

In another aspect, the disclosure relates to a method of treating or preventing or reducing the severity of an infectious disease in a subject, e.g., by inducing an immune response against the infectious disease in the subject. In some aspects, the method includes administering a priming composition comprising an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein, and administering a booster composition comprising an effective amount of the RNA molecules, RNA-LNPs and/or compositions. In some aspects, the composition elicits an immune response including an antibody response. In some aspects, the composition elicits an immune response including a T cell response.

In another aspect, the disclosure relates to a method of treating or preventing or reducing the severity of a VZV infection and/or disease caused by VZV in a subject, e.g., by inducing an immune response to VZV in the subject. In some aspects, the method includes administering a priming composition comprising an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein, and administering a booster composition comprising an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein. In some aspects, the composition elicits an immune response including an antibody response. In some aspects, the composition elicits an immune response including a T cell response.

The methods disclosed herein may involve administering to a subject a VZV RNA-LNP composition comprising at least one VZV RNA molecule having an open reading frame encoding at least one VZV antigenic polypeptide, thereby inducing an immune response in the subject specific to the VZV antigenic polypeptide, wherein anti-antigen polypeptide antibody titers in the subject are increased following vaccination compared to anti-antigen polypeptide antibody titers in subjects vaccinated with a prophylactically effective dose (e.g., a therapeutically effective dose that prevents infection with the virus at a clinically acceptable level) of a traditional vaccine against VZV. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide. In some embodiments, the anti-antigen polypeptide antibody titer in the subject is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 log, after administration of the VZV RNA-LNP composition, compared to the anti-antigen polypeptide antibody titer in a subject administered a prophylactically effective dose of a traditional composition against VZV.

The methods disclosed herein may involve administering to a subject a VZV RNA-LNP composition comprising at least one VZV RNA molecule having an open reading frame encoding at least one VZV antigenic polypeptide, thereby inducing an immune response in the subject specific to the VZV antigenic polypeptide, wherein the immune response in the subject is at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 75, 80, 82, 84, 86, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153, 154, 155, 156, 158, 1 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times, up to 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 , 96, 98, or 100 times, or between any two of, or exactly 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times , 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times the dosage level of a traditional composition against VZV.

In some embodiments, the RNA molecules, RNA-LNPs and/or compositions are used as vaccines. In some embodiments, the RNA molecules, RNA-LNPs and/or compositions may be used in a variety of therapeutic or prophylactic methods to prevent, treat or ameliorate herpes zoster or shingles, or disorders related to herpes zoster or shingles.

In some embodiments, the RNA molecules, RNA-LNPs and/or compositions may be used in a variety of therapeutic or prophylactic methods for preventing, treating or ameliorating post-herpetic neuralgia.

The VZV RNA composition may be administered prophylactically or therapeutically to healthy subjects, or early in the infection during the latent period or during active infection after the onset of symptoms. In some embodiments, the subject is immunocompetent. In some embodiments, the subject is immunocompromised.

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

In some embodiments, the RNA molecules, RNA-LNPs and/or compositions are administered intramuscularly (IM) or intradermally (ID).

The present disclosure further provides kits comprising the RNA molecules, RNA-LNPs, and/or compositions.

In some embodiments, the RNA molecules, RNA-LNPs and/or compositions described herein are administered to subjects less than about 1 year of age, or between about 1 and about 10 years of age, or between about 10 and about 20 years of age, or between about 20 and about 50 years of age, or between about 60 and about 70 years of age, or older.

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

In some embodiments, the subject is at least 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, up to 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, exactly 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 between any two of 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. In some embodiments, the subject may be about 50 years or older.

In some embodiments, the subject is at least 1 year or older, 5 years or older, 10 years or older, 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, up to 1 year or older, 5 years or older, 10 years or older, 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, or up to exactly 1 year older, 5 years or older, 10 years or older, 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older or older, 5 or older, 10 or older, 20 or older, 30 or older, 40 or older, 50 or older, 60 or older, 70 or older, or between any two of 1 or older, 5 or older, 10 or older, 20 or older, 30 or older, 40 or older, 50 or older, 60 or older, 70 or older, or older. In some embodiments, the subject may be 50 years old or older.

X. Clinical Studies The VZV RNA-LNP vaccine of the present disclosure comprises nucleoside-modified mRNA (modified RNA; modRNA) encoding glycoprotein E (gE) from VZV. The VZV RNA-LNP vaccine may comprise RNA comprising a single-stranded, 5'-capped and polyadenylated modified RNA that is translated after entering the cell. The RNA comprises an open reading frame (ORF) encoding a variation of VZV gE. For example, the RNA molecule may comprise gE_WT CO2 (RNA encodes a full-length gE protein that is localized in the plasma membrane and Golgi), gE ms5 CO1 (RNA encodes a truncated gE protein at the C-terminus that is primarily localized in the plasma membrane) and/or gE ms6 CO2 (RNA encodes the ectodomain of the secreted gE protein). Additionally, as described herein, the RNA may comprise structural elements optimized for high efficacy of the RNA, such as untranslated regions (UTRs). VZV RNA-LNPs may include RNA as provided in Table 5 of Example 1 disclosed herein. VZV RNA-LNPs may include RNA as provided in Tables 1-3 of Example 7 disclosed herein. The RNA may also include substitution of uridine with 1-methyl-pseudouridine to decrease recognition of the vaccine RNA by innate immune sensors, such as toll-like receptors (TLR) 7 and 8, resulting in decreased innate immune activation and increased protein translation.

The RNA molecules described herein are formulated/encapsulated in lipid nanoparticles (LNPs) to enable delivery of the RNA into host cells following intramuscular (IM) injection. The LNP formulation may include two functional lipids, ALC-0315 and ALC-0159, and two structural lipids, DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol. The efficacy of RNA vaccines is optimized by LNP encapsulation, which protects the RNA from degradation by extracellular RNases and facilitates delivery in cells. Following IM injection of the VZV RNA-LNP vaccine, the LNPs are taken up by cells and the RNA is released into the cytosol, where the RNA is translated and the encoded viral antigens are produced.

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

The clinical study of the present disclosure evaluates the safety, tolerability, and immunogenicity of the VZV RNA-LNP vaccine against VZV. For example, the VZV RNA-LNP vaccine may be applied for active immunization for the prevention of varicella-zoster disease caused by VZV for adults (e.g., 45 years or older, 50 years or older, 55 years or older, 60 years or older, 70 years or older, etc., or 50-69 years old). The VZV RNA-LNP vaccine may be administered in different dose levels, dose formulations, number of doses, and dosing schedules as described herein, including, but not limited to:
- a single dose schedule or a two dose schedule (e.g. day 0 and about 2 months later or day 0 and about 6 months later)
- different dose levels (e.g., about 15 μg, about 30 μg, about 60 μg, about 90 μg, about 100 μg or higher dose levels per administration);
- Different formulations (non-lyophilized and/or lyophilized).

VZV RNA-LNPs may be presented as liquid or lyophilized formulations. Administration of VZV RNA-LNP vaccines may be dosed in the range of about 15 μg, about 30 μg, about 60 μg, about 90 μg, about 100 μg or higher per dose in an injection volume of about 0.25-1 mL (e.g., about 0.25, 0.5, 1 mL). Dilution with sterile 0.9% sodium chloride (normal saline) may be required.

The objectives of VZV RNA-LNP clinical studies may include, but are not limited to, the following:
- To describe the safety and tolerability profile of the VZV RNA-LNP vaccine administered at selected dose levels and schedules in participants.
- To describe the immune responses elicited by SHINGRIX® and VZV RNA-LNP vaccines administered at selected dose levels and schedules in participants.

Below are examples of specific modes for carrying out the present disclosure. The following examples are included to demonstrate aspects of the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. It should be understood by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to work well in carrying out the present disclosure. However, those of skill in the art should understand in light of this disclosure that many changes can be made in the specific modes disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be taken into account.

Example 1
Generation of VZV RNA Constructs The RNA constructs generated herein encode VZV gE wild type (WT) and gE variant proteins with cytoplasmic tail (CT) and/or transmembrane (TM) domain modifications. Figure 1 and Table 4 show the WT gE protein (gE WT), variant gE proteins with cytoplasmic tail modifications (ms4, ms5, ms8, ms9, ms10, ms11, and ms12), and variant gE proteins with TM modifications (ms3 and ms6).

DNA sequences encoding VZV proteins were prepared and utilized for in vitro transcription reactions to generate RNA. In vitro transcription of RNA is known in the art and described herein. DNA templates were cloned into plasmid vectors with backbone sequence elements (T7 promoter, 5' and 3' UTR, polyA tail for RNA stability and improved translation efficiency). DNA was purified, quantified spectrophotometrically, and in vitro transcribed with T7 RNA polymerase in the presence of trinucleotide Cap 1 analog (( _m27,3' ^-O )Gppp( ^m2'-O )ApG) (TriLink) and with N1-methylpseudouridine (Ψ) replacing uridine (modified RNA; modRNA).

VZV RNA was generated from DNA that was codon optimized (CO) for stabilization and superior protein expression. As used herein, CO1 indicates a G/C content of about 58%, CO2 indicates a G/C content of about 66%, and CO3 indicates a G/C content of about 62%. Table 5 shows the RNA constructs of the present disclosure, including the 5'UTR, open reading frame encoding a Varicella Zoster Virus (VZV) polypeptide, 3'UTR, and polyA tail, and the corresponding sequences.

The VZV RNA constructs/molecules and RNA-LNPs evaluated in the in vitro and in vivo experiments described in the Examples herein contain modified RNA (modRNA) that contains an RNA sequence in which all uridines are replaced by N1-methylpseudouridine (Ψ).

Example 2
Expression and subcellular location of VZV gE (Vero cells)
Expression of VZV RNA constructs was tested in transfected Vero cells, a kidney epithelial cell culture line derived from African green monkeys. Seeded Vero cells were transfected with 10 ng, 25 ng, or 50 ng of RNA constructs (Table 5) for 24 hours at 37° C., 5% CO ₂ using MESSENGERMAX™ according to the manufacturer's instructions. Cells were washed three times with PBS+Ca/Mg and fixed in 4% paraformaldehyde (PFA) for 20 minutes at 25° C. Cells were washed twice with 3% bovine serum albumin (BSA) in PBS+Ca/Mg. Primary and secondary staining antibodies were diluted in 0.1% saponin in goat serum. Cells were stained with a 1:1000 dilution of primary antibody for 1 hour at 37° C., washed 3 times with PBS+Ca/Mg, and incubated with secondary antibody (1:500) and CELLMASK™ (1:140,000) for 45 minutes at 37° C. Cells were then stained with Dapi (1:15,000-1:20,000) for 15 minutes at 25° C. and washed 3 times with PBS+Ca/Mg. Cells were analyzed for VZV gE expression and subcellular localization using the Opera PHENIX® Plus High-Content Screening System at 10x or 63x magnification.

Imaging analysis revealed an RNA dose-dependent increase in the percentage of VZV gE ⁺ transfected Vero cells, with nearly 100% of cells expressing VZV gE at the 50 ng dose among the cytoplasmic tail mutants (Figure 2) and approximately 80% of cells expressing VZV gE at the 50 ng dose among the secreted mutants (Figure 3). The secreted mutants are primarily transported to the outside of the cell, with lower levels detected inside.

The mean fluorescence intensity (MFI) of each transfected RNA construct reveals a dose-dependent increase in VZV gE expression among the cytoplasmic tail mutants (Figure 4) and the secreted mutants (Figure 5), as well as a positive correlation between higher G/C content (CO2) and higher VZV gE expression levels among the cytoplasmic tail mutants (Figure 4).

Imaging analysis reveals the subcellular localization of VZV gE in Vero cells transfected with various RNA constructs. Localization of VZV gE in gE_WT transfected cells was in the plasma membrane and trans-Golgi network (TGN) (Fig. 6, 63x magnification; Fig. 10, 10x magnification). Cytoplasmic tail mutants (ms4, ms5, ms8, ms9, ms10, ms11, ms12) showed VZV gE localization preferentially in the plasma membrane (Figs. 6-8, 63x magnification; Figs. 10-12, 10x magnification). Secreted mutants (ms3, ms6) showed VZV gE localization in the culture supernatant (Fig. 9, 63x magnification; Fig. 13, 10x magnification).

Example 3
Preparation of VZV gE modRNA Formulated in LNPs Purified RNA (described in Table 5) was formulated/encapsulated in lipid nanoparticles (RNA-LNPs) using an ethanol lipid mixture of ionizable cationic lipids and transferred via diafiltration into an aqueous buffer system to obtain lipid nanoparticle compositions as described herein. RNA-LNPs contain VZV RNA molecules, a cationic lipid, ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), a PEGylated lipid, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide, and two structural lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC] and cholesterol. See Table 6.

Example 4
In vitro expression (HEK 293T cells)
An in vitro expression (IVE) analysis was performed to evaluate the efficacy of the VZV RNA-LNP vaccine. RNA constructs were formulated in LNPs as previously described in Example 3, and a range of input VZV RNA-LNP amounts were transfected into human embryonic kidney (HEK) 293T cells for 24 hours at 37°C, 5% _CO2 . Transfected cells were washed with DPBS, detached from plate wells using ACCUTASE®, and rinsed with cold PBS. Cells were subjected to live/dead staining, fixation, and permeabilization prior to FACS staining. Fixed and permeabilized cells were incubated with anti-gE primary antibody (1:1000) for 45 minutes at 2-8°C, washed twice with BD PERM/WASH™ buffer, and incubated with goat anti-human kappa-PE secondary antibody (1:1000) for 30 minutes at 2-8°C. Cells were washed twice with BD PERM/WASH™ buffer, resuspended in 1x FACS buffer, and analyzed on a BD LSRII to determine the percentage of cells expressing VZV gE for each VZV RNA-LNP input. Titration curves were generated in which the amount of input RNA was plotted against the percentage of gE-expressing cells to determine the EC50 of each VZV RNA-LNP vaccine.

The data in Table 7 and Figure 14 show a positive correlation between VZV RNA-LNP vaccine potency (e.g., lower EC50) and higher MFI. Comparison of MFI of VZV RNA-LNP (293T cells) and VZV gE RNA (Vero cells) (see Example 2) showed that both VZV RNA-LNP and VZV gE RNA shared similar MFI trends (Table 7), with potency and MFI of gE_WT CO2 > gE_WT CO1 > ms4 CO1 > ms3 CO1.

These trends were also observed for the data in Table 8 and Figure 15, where potency and MFI were ms5 CO1 > gE_WT CO2 > gE_WT CO1 > ms6 CO2.

(Example 5)
Immune response (in vivo experiments)
The VZV RNA-LNP vaccine was tested for its ability to induce IgG and T cell responses in Balb/c mice. 15 mice per group were immunized according to the schedule and specifications in Table 9. Briefly, 15 mice per group were immunized intramuscularly (IM) on day 0 and boosted IM on day 28. Mice immunized with SHINGRIX® received 1, 2.5, or 5 μg, corresponding to 1/50, 1/20, and 1/10 of the human clinical dose (50 μg), respectively; mice immunized with gE_WT CO1 received 0.5 or 1 μg; mice immunized with ms3 CO1 received 0.5 μg; mice immunized with ms4 CO1 received 0.5 μg; mice immunized with gE_WT CO2 received 0.5 μg; mice immunized with lyophilized gE_WT CO1 received 0.5 or 1 μg. Saline was administered to the negative control groups on days 0 and 28. Serum was collected from mice on days 28 and 42 for characterization of VZV gE-binding IgG levels (LUMINEX® analysis). Spleens were harvested and serum was collected from five mice in each group on day 35 for cellular analysis.

IgG antibody titers Serum IgG levels were determined for each mouse sample using the LUMINEX® platform. Briefly, diluted sera were incubated in the dark in the presence of blocked recombinant gE protein-binding singleplex microspheres (MAGPLEX® Microspheres, region 79) for 20±4 hours at 2-8°C, 300 rpm. Secondary antibody solution (R-phycoerythrin Conjugated AffiniPure F(ab)'2 Fragment Goat Anti-Mouse IgG F(ab)'2 Fragment Specific) was prepared at 1:250 in LXA-16 buffer. Plates were washed 3 times in LXA-20 (EIA-7) and secondary antibody was applied to each well of the assay plate. Plates were covered with aluminum sealers and incubated for 2 hours ± 15 minutes at 25°C, 300 rpm. Plates were washed 3 times with LXA-20 (EIA-7) buffer and 100 ul of LXA-20 was applied to each well. Plates were covered with aluminum sealers and incubated for a minimum of 4 minutes or a maximum of 2 hours at 25°C, 300 rpm. Plates were analyzed on a BIO-PLEX® Reader. IgG titers on days 28, 34 and 42 are shown in Table 10 and Figures 17-19.

As shown in Figure 16, IgG titers at day 28 (prime only/before boost) reveal dose-dependent changes in geometric mean concentrations (GMCs) in mice given SHINGRIX®, gE_WT CO1, and lyophilized gE_WT CO1. The GMCs of gE_WT CO1 (7.69) and gE_WT CO2 (12.90) administered at 0.5 μg (1/60 of the potential clinical dose of 30 μg) are significantly higher than the GMC of SHINGRIX® (2.33) administered at 1 μg (1/50 of its human clinical dose of 50 μg) (Figure 16). Potential clinical doses for VZV RNA-LNP include, but are not limited to, 15 μg, 30 μg, 60 μg, 90 μg, 100 μg, or higher doses. Thus, the GMC of gE_WT CO1 administered at 1 μg (1/60 of the potential clinical dose of 60 μg) (33.78) is higher than the GMC of SHINGRIX® administered at 1 μg (1/50 of its human clinical dose of 50 μg) (2.33).

As shown in Figure 17, IgG titers on day 34 (6 days after the boost on day 28) similarly reveal dose-dependent changes in GMC in mice given SHINGRIX®, gE_WT CO1, and lyophilized gE_WT CO1 (Figure 17). Higher IgG levels were observed for the VZV RNA-LNP vaccine compared to SHINGRIX® (notably, GMCs of 223.9 for gE_WT CO1 and 437.9 for gE_WT CO2 at 1/60 of the potential clinical dose of 30 μg, versus 177.9 for SHINGRIX® at 1/50 of its human clinical dose (Figure 17)).

A positive correlation was observed between higher G/C content and higher IgG titers when comparing gE_WT CO1 (approximately 58% G/C) and gE_WT CO2 (approximately 66% G/C). See Figures 16 and 17.

As shown in Figure 18, IgG titers at day 42 further reveal a dose-dependent change in geometric mean concentration (GMC). Higher IgG levels were observed for gE_WT CO1 (GMC of 918.43) and gE_WT CO2 (GMC of 524.39) compared to SHINGRIX® (GMC of 498.79).

Figure 19 shows a summary of IgG levels observed for SHINGRIX® (1 μg) and RNA-LNP vaccine (0.5 μg) on days 28 (prime only), 34 (6 days after boost), and 42.

Comparable IgG titers were observed for non-lyophilized gE_WT CO1 and lyophilized gE_WT CO1 Lyo.

Cell-mediated immunity (T cell response)
Spleen cells were harvested from Balb/c mice on day 34 (34 days after immunization and 6 days after boost) to assess the induced gE-specific T cell responses. Intracellular cytokine staining (ICS) assays were performed. The ICS assay was used to detect the presence of cytokines in CD4 ⁺ or CD8 ⁺ T cells after antigen peptide stimulation. The ICS assay detects cytokines produced in both CD4 ⁺ and CD8 ⁺ T cells after antigen peptide stimulation. Multiple cytokines can be detected, including IFN-γ. During ex vivo stimulation of splenocytes, reagents to block protein secretion are added, retaining the cytokines synthesized and allowing intracellular staining. After stimulation, cells were stained for surface and intracellular markers to identify T cell type (CD3 ⁺ cells for CD4 and CD8 T cells), activation markers (CD154/CD40L) and cytokines. Identify. gE-specific T cells were assessed by assessing CD4 ⁺ T cells expressing IFN-γ, IL-2, TNFα and CD40L, and CD8+ T cells expressing IFN-γ.

^2x106 splenocytes were stimulated with a mix of 2μg/mL gE peptide pool mix, 10ng/ml phorbol myristate acetate (PMA) and 1μg/mL ionomycin (positive control), or DMSO (negative control). BD GOLGISTOP™ and BD GOLGIPLUG™ were added to block protein secretion. After 6 hours of incubation at 37°C, cells were stained for viability markers (10 min at 25°C) and extracellular markers (20 min at 25°C) using directly labeled antibodies. Cells were fixed and permeabilized with BD CYTOFIX/CYTOPERM™ solution. Intracellular staining for cytokines (IFN-γ, IL-2, TNFα) and activation markers (CD154/CD40L) was performed in BD CYTOFIX/CYTOPERM™ solution (30 min at 25° C.). Cells were washed and resuspended in 2% FBS/PBS buffer and acquired on an LSRFORTESSA™. Data was analyzed with FlowJo 10.7.1. Results shown are background (medium-DMSO) subtracted.

Examination of CD4 ⁺ IFN-γ ⁺ (Th1) T cells revealed strong, dose-dependent responses in groups given the VZV RNA-LNP vaccine (including lyophilized vaccine) and minimal responses in mice given SHINGRIX®, as shown in Figure 20. Examination of CD8 ⁺ IFN-γ ⁺ T cells revealed strong but variable responses in groups given the VZV RNA-LNP vaccine and undetectable responses in mice given SHINGRIX®, as shown in Figure 21.

Example 6
Immune response – LAV-experienced mice (in vivo experiments)
The VZV RNA-LNP vaccine was tested for its ability to induce IgG and T cell responses in C57BL/6 mice. 15 mice per group were immunized according to the schedule and specifications in Table 11. Briefly, 10 or 15 mice per group were primed subcutaneously (SQ) with live attenuated varicella (LAV) vaccine (VARIVAX®, Merck) on day 0 using a full human dose (1350 pfu) per mouse, immunized intramuscularly (IM) on day 35, and boosted on day 63. "Infection" with the LAV vaccine mimics the exposure to VZV given to humans when they become infected with VZV as children and develop chicken pox.

Mice immunized with SHINGRIX® received 1, 2.5, or 5 μg, corresponding to 1/50, 1/20, and 1/10 of the human clinical dose, respectively; mice immunized with gE_WT CO2 received 0.5 or 1 μg VZV RNA-LNP; mice immunized with ms5 CO1 received 0.5 or 1 ug VZV RNA-LNP; mice immunized with ms6 CO2 received 0.5 or 1 ug VZV RNA-LNP; mice immunized with lyophilized gE_WT CO2 received 0.5 or 1 ug VZV RNA-LNP. Saline was administered to the negative control groups on days 35 and 63. Serum was collected from mice on days 35, 63, and 76 for characterization of VZV gE-binding IgG levels (LUMINEX® analysis). Spleens were harvested on day 48 (13 days after dose 1 for selected groups) and day 76 for cellular analysis (T-cell and B-cell responses).

IgG Antibody Titers Serum IgG levels were determined for each mouse sample using the LUMINEX® platform (described herein). IgG titers for days 35, 63 (1 month after dose 1) and 76 (13 days after dose 2) are shown in Table 12 and Figures 22-24. Each data point in the figures is the result from an individual animal; each horizontal line represents the geometric mean IgG concentration (μg/ml) and the whiskers represent the 95% confidence interval.

As expected prior to immunization, FIG. 22 shows low levels of IgG titers at day 35 after SQ prime with LAV vaccine (VARIVAX®) on day 0. As shown in FIG. 23, IgG titers increased significantly in a dose-dependent response at day 63 (1 month after dose 1) in LAV-experienced mice given VZV RNA-LNP vaccine or SHINGRIX®. FIG. 24 shows a further increase in IgG titers in a dose-dependent response at day 76 (13 days after dose 2/boost).

The effect of lyophilization on immunogenicity was evaluated. As shown in Figure 25, comparable IgG titers were observed for non-lyophilized gE_WT CO2 and lyophilized gE_WT CO2 at both day 63 (1 month after dose 1) and day 76 (13 days after dose 2/boost).

Cell-Mediated Immunity (T-Cell and B-Cell Responses)
Vaccine-induced T cell responses were measured by ELISpot and intracellular cytokine staining (ICS) assays (described herein) after ex vivo stimulation of splenocytes with gE peptide pools (2 μg/mL of each peptide). For ELISpot, the cytokine IFN-γ secreted by activated T cells was captured by an anti-IFN-γ antibody coated on a polyvinylidene difluoride (PVDF) membrane at the bottom of the wells on a microplate. The captured IFN-γ was developed into spots by another non-competitive biotinylated anti-IFN-γ secondary antibody, followed by an enzyme color reaction using streptavidin-alkaline phosphatase (ALP) conjugate and substrate solution, nitroblue tetrazolium and 5-bromo-4-chloro-3′-indolyl phosphate (BCIP/NBT-plus), which results in a dark purple precipitate or spot. T cell IFN-γ responses were measured using the Mabtech Mouse IFN-γ ELISpot PLUS kit (ALP) and expressed as spot forming cells (SFC) per million cells. IFN-γ expressing cells in CD4 ⁺ and CD8 ⁺ T cells, expressed as the percentage of IFN-γ ⁺ cells in CD4 ⁺ and CD8 ⁺ T cells, were measured by ICS assay.

Vaccine-induced B cell responses were assessed by measuring the frequency of VZV gE-specific B cells in the spleen. Wild-type gE protein ectodomain with streptag was coupled to streptavidin (SA)-fluorochrome PE and APC, both from BioLegend, in a 2:1 ratio in separate tubes at 20x the desired staining concentration (10 μg/mL for each protein) for 1 hour at room temperature (RT). Each of the SA-fluorochrome-coupled spike proteins was pooled and diluted 1x using flow cytometry (FC) buffer (2% FBS/PBS) to generate B cell probes for identifying gE-specific B cells. Single cell suspensions of spleen cells ( ^5x106 cells/well) were first washed in PBS and stained with eFluor 506 Fixable Viability dye for 10 minutes at RT to identify live from dead cells. After washing in FC buffer, cells were incubated with 50 μL/well of 1× B cell probe for 30-45 min and then washed to remove unbound probe. Cells were surface stained with a cocktail of flow cytometry antibodies (CD19, IgD, IgM, IgD, all from BioLegend) to identify B cell surface phenotype. After washing, cells were fixed using BD fixation buffer and suspended in FC buffer. Cells were acquired on an LSR Fortessa and data were analyzed by FlowJo (10.7.1). gE-specific B cell results were expressed as the percentage of IgG-expressing B cells.

Day 48 Spleen cells were harvested from C57BL/6 mice on day 48 (13 days after dose 1) to evaluate gE-specific cellular immune responses induced after a single vaccine dose (see Table 13 and Figures 26A-26D). LAV-experienced mice were immunized IM with SHINGRIX® or VZV RNA-LNP vaccines on day 35 and spleens were harvested on day 48 (13 days after dose 1).

Figure 26A shows that LAV-experienced mice elicited strong gE-specific IFN-γ (T cell) responses in a dose-dependent manner as measured by IFN-γ ELISpot after a single vaccine dose. As shown in Figure 26B, the results of the ICS assay revealed strong, dose-dependent gE-specific IFN-γ ⁺ CD4 ⁺ T cell responses induced by the vaccine, similar to those demonstrated by ELISpot. Figure 26C shows that gE-specific IFN-γ ⁺ CD8 ⁺ T cell responses were induced only by the VZV RNA-LNP vaccines (gE_WT CO2, ms5 CO1, ms6 CO2, gE_WT CCO2 lyo), but not by SHINGRIX®, demonstrating the unique immune response induced by the VZV-RNA-LNP vaccine. Overall, on day 48 (13 days after dose 1), T cell responses induced by 1 μg (1/60 of the potential clinical dose of 60 μg) of gE_WT CO2 were significantly higher than those induced by 1 μg (1/50 of its human dose) of SHINGRIX®.

B cell responses were assessed in splenocytes by measuring ^{the frequency of gE-specific IgG + B} cells by flow cytometry, which revealed that the vaccine-induced B cell responses were similar to the gE-specific IFN-γ ⁺ CD4 ⁺ T cell responses, as shown in Figure 26D.

Day 76 Spleen cells were harvested from C57BL/6 mice on day 76 (13 days after dose 2/boost) to evaluate gE-specific cellular immune responses induced after the second/boost vaccine dose (see Table 14 and Figures 27A-27C). LAV-experienced mice were immunized IM with SHINGRIX® or VZV RNA-LNP vaccines on days 35 and 63 and spleens were harvested on day 76 (13 days after dose 2/boost).

Figure 27A shows that the second/boost vaccine dose significantly increased gE-specific CD4 ⁺ T cell responses in a dose-dependent manner as measured by ICS assay. As shown in Figure 27B, robust increases in gE-specific IFN-γ ⁺ CD8 ⁺ T cell responses were induced only by VZV RNA-LNP vaccines (gE_WT CO2, ms5 CO1, ms6 CO2, gE_WT CCO2 lyo) but not by SHINGRIX®, confirming the unique immune response induced by the VZV-RNA-LNP vaccine. Overall, on day 76 (13 days after dose 2/boost), T cell responses induced by 1 μg (1/60 of the potential clinical dose of 60 μg) of gE_WT CO2 were higher than those induced by 1 μg (1/50 of its human dose) of SHINGRIX®.

B cell responses were assessed in splenocytes by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry and are shown in FIG. 27C.

(Example 7)
VZV Antigens The sequences of the VZV antigens/polypeptides, VZV DNA and VZV RNA of the present invention are provided in Tables 1-3. The sequences may include any stop codon, including but not limited to those provided in the tables.

The following paragraphs describe additional aspects of the present disclosure:

1. An RNA molecule comprising at least one open reading frame encoding a varicella zoster virus (VZV) polypeptide.

2. The RNA molecule of paragraph 1, wherein the VZV polypeptide is a VZV glycoprotein.

3. The RNA molecule of paragraph 2, wherein the VZV glycoprotein is selected from VZV gK, gN, gC, gB, gH, gM, gL, gI and gE.

4. The RNA molecule of paragraph 3, wherein the VZV glycoprotein is VZV gE.

5. The RNA molecule of any one of paragraphs 1 to 4, wherein the VZV polypeptide is full-length, truncated, fragment or variant thereof.

6. An RNA molecule described in any one of paragraphs 1 to 5, wherein the VZV polypeptide comprises at least one mutation.

7. The RNA molecule of any one of paragraphs 1 to 6, wherein the VZV polypeptide comprises an amino acid sequence of Table 1, including, but not limited to, any of SEQ ID NOs: 1 to 11.

8. The RNA molecule of any one of paragraphs 1 to 7, wherein the VZV polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NOs: 1 to 11.

9. The RNA molecule of any one of paragraphs 1 to 8, wherein the VZV polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 11.

10. An RNA molecule according to any one of paragraphs 1 to 9, wherein the open reading frame is transcribed from a nucleic acid sequence in Table 2, including, but not limited to, any of SEQ ID NOs: 12 to 145.

11. An RNA molecule according to any one of paragraphs 1 to 10, 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 SEQ ID NOs: 12 to 145.

12. An RNA molecule according to any one of paragraphs 1 to 11, wherein the open reading frame comprises a nucleic acid sequence of Table 3, including, but not limited to, any of SEQ ID NOs: 146 to 279.

13. An RNA molecule according to any one of paragraphs 1 to 12, 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 SEQ ID NOs: 146 to 279.

14. An RNA molecule described in any one of paragraphs 1 to 13, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 146 (gE WT).

15. An RNA molecule described in any one of paragraphs 1 to 14, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 147 (gE WT CO1).

16. An RNA molecule described in any one of paragraphs 1 to 15, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 148 (gE WT CO2).

17. An RNA molecule according to any one of paragraphs 1 to 16, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 149 (ms3 CO1).

18. An RNA molecule described in any one of paragraphs 1 to 17, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 150 (ms3 CO2).

19. An RNA molecule according to any one of paragraphs 1 to 18, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 151 (ms4 CO1).

20. An RNA molecule described in any one of paragraphs 1 to 19, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 152 (ms4 CO2).

21. An RNA molecule according to any one of paragraphs 1 to 20, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 153 (ms5 CO1).

22. An RNA molecule according to any one of paragraphs 1 to 21, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 154 (ms5 CO2).

23. An RNA molecule according to any one of paragraphs 1 to 22, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 155 (ms5 CO2 v2).

24. An RNA molecule according to any one of paragraphs 1 to 23, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 156 (ms6 CO1).

25. An RNA molecule according to any one of paragraphs 1 to 24, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 157 (ms6 CO2).

26. An RNA molecule according to any one of paragraphs 1 to 25, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 158 (ms8 CO1).

27. An RNA molecule according to any one of paragraphs 1 to 26, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 159 (ms9 CO1).

28. An RNA molecule described in any one of paragraphs 1 to 27, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 160 (ms9 CO2).

29. An RNA molecule according to any one of paragraphs 1 to 28, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 161 (ms10 CO1).

30. An RNA molecule according to any one of paragraphs 1 to 29, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 162 (ms10 CO2).

31. An RNA molecule according to any one of paragraphs 1 to 30, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 163 (ms10 CO3).

32. An RNA molecule according to any one of paragraphs 1 to 31, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 164 (ms11 CO1).

33. An RNA molecule according to any one of paragraphs 1 to 32, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 165 (ms11 CO2).

34. An RNA molecule described in any one of paragraphs 1 to 33, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 166 (ms12 CO1).

35. An RNA molecule according to any one of paragraphs 1 to 34, wherein the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 167 (ms12 CO2).

36. An RNA molecule according to any one of paragraphs 1 to 35, wherein each uridine is replaced by N1-methylpseudouridine (Ψ).

37. An RNA molecule according to any one of paragraphs 1 to 36, further comprising a 5' untranslated region (5'UTR).

38. The RNA molecule of paragraph 37, wherein the 5'UTR comprises a sequence selected from any one of SEQ ID NOs: 281 and 312-313.

39. An RNA molecule according to any one of paragraphs 1 to 38, further comprising a 3' untranslated region (3'UTR).

40. The RNA molecule of paragraph 39, wherein the 3'UTR comprises a sequence selected from any of SEQ ID NOs: 284, 314 and 317.

41. An RNA molecule according to any one of paragraphs 1 to 40, comprising a 5' cap portion.

42. The RNA molecule of paragraph 41, comprising a 5' cap portion comprising (3'OMe) _-m27,3' ^- OGppp( _m12' ^-O )ApG.

43. The RNA molecule of any one of paragraphs 1 to 42, further comprising a 3' polyA tail.

44. The RNA molecule of paragraph 43, wherein the polyA tail comprises a sequence selected from any of SEQ ID NOs: 287 and 315, comprising +/-1 or +/-2 adenosines (A).

45. The RNA molecule of paragraph 43, wherein the polyA tail comprises the sequence of SEQ ID NO:287, which contains +/-1 or +/-2 adenosines (A).

46. The RNA molecule of paragraph 43, wherein the polyA tail comprises the sequence of SEQ ID NO: 315, including +/- 1 or +/- 2 adenosines (A).

47. An RNA molecule described in any one of paragraphs 1 to 46, comprising a 5'UTR and a 3'UTR.

48. An RNA molecule described in any one of paragraphs 1 to 47, comprising a 5' cap, a 5' UTR, and a 3' UTR.

49. An RNA molecule according to any one of paragraphs 1 to 48, comprising a 5' cap, a 5' UTR, a 3' UTR, and a poly A tail.

50. An RNA molecule according to any one of paragraphs 1 to 49, comprising a 5'UTR comprising the sequence of SEQ ID NO: 281 or 312, an open reading frame comprising the sequence of any one of SEQ ID NOs: 146 to 279, and a 3'UTR comprising the sequence of SEQ ID NO: 284 or 317.

51. An RNA molecule according to any one of paragraphs 1 to 50, comprising a 5'UTR comprising any one of the sequences of SEQ ID NOs: 28, 312 and 313, an open reading frame comprising any one of the sequences of SEQ ID NOs: 146-279, a 3'UTR comprising any one of the sequences of SEQ ID NOs: 284, 314 and 317, and a polyA tail comprising any one of the sequences of SEQ ID NOs: 287 and 315.

52. An RNA molecule according to any one of paragraphs 1 to 51, wherein the open reading frame is generated from codon-optimized DNA.

53. An RNA molecule according to any one of paragraphs 1 to 52, wherein the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, about 50%-75%, about 55%-70%, about 58%, about 66% or about 62%.

54. An RNA molecule according to any one of paragraphs 1 to 53, wherein the encoded VZV polypeptide is localized in the cell membrane, localized in the Golgi, and/or anchored in the membrane and secreted.

55. An RNA molecule according to any one of paragraphs 1 to 54, comprising stabilized RNA.

56. An RNA molecule according to any one of paragraphs 1 to 55, wherein the RNA comprises at least one modified nucleotide.

57. The RNA molecule of paragraph 56, 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-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine.

58. The RNA molecule of paragraph 56 or 57, wherein the modified nucleotide is N1-methylpseudouridine (Ψ).

59. An RNA molecule according to any one of paragraphs 1 to 58, wherein each uridine is replaced by N1-methylpseudouridine (Ψ).

60. The RNA molecule of any one of paragraphs 1 to 59, wherein the RNA is an mRNA or a self-replicating RNA.

61. The RNA molecule of paragraph 60, wherein the RNA is mRNA.

62. A composition comprising an RNA molecule according to any one of paragraphs 1 to 61, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

63. The composition of paragraph 62, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEGylated lipid, and at least a first and a second structural lipid.

64. The composition of paragraph 62 or 63, wherein the lipid nanoparticles comprise a cationic lipid.

65. The composition of paragraph 64, wherein the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315).

66. The composition of any one of paragraphs 61 to 64, wherein the lipid nanoparticles comprise a PEGylated lipid.

67. The PEGylated lipid is a glycol lipid including 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-ditetradecylacetamide, PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), and PEG-2000-DMG, PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy- 67. The composition according to any one of paragraphs 63 to 66, wherein the PEG is 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), 4-O-(2',3'-di(tetradecanoyloxy)propyl)-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate.

68. The composition of any one of paragraphs 63 to 67, wherein the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

69. The composition of any one of paragraphs 63 to 68, wherein the first structural lipid is a neutral lipid.

70. The neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine (DOPE). The composition according to paragraph 69, which is diolamine 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-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

71. The composition of paragraph 69 or 70, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

72. The composition of any one of paragraphs 63 to 71, wherein the second structural lipid is a steroid or a steroid analog.

73. The composition of paragraph 72, wherein the steroid or steroid analog is cholesterol.

74. The composition of any one of paragraphs 62 to 73, wherein the lipid nanoparticles have an average diameter of about 1 to about 500 nm.

75. The composition of any one of paragraphs 62 to 74, comprising an RNA molecule at a concentration of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of about 0.8 to 0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05 to 0.15 mg/mL, a neutral lipid at a concentration of about 0.1 to 0.25 mg/mL, and a steroid or steroid analog at a concentration of about 0.3 to 0.45 mg/mL.

76. The composition of any one of paragraphs 62 to 75, comprising an RNA molecule at a concentration of about 0.01 to 0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315) at a concentration of about 0.8 to 0.95 mg/mL, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159) at a concentration of about 0.05 to 0.15 mg/mL, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of about 0.1 to 0.25 mg/mL, and cholesterol at a concentration of about 0.3 to 0.45 mg/mL.

77. The composition of any one of paragraphs 62 to 76, comprising an RNA molecule formulated in a lipid nanoparticle (LNP) at a concentration of about 0.06 mg/mL.

78. The composition of any one of paragraphs 62 to 77, further comprising at least one of a buffer, a stabilizer, a salt, a surfactant, a preservative, an excipient, or an adjuvant.

79. The composition of any one of paragraphs 62 to 78, further comprising at least a buffer and a stabilizer, and optionally a salt diluent.

80. The composition of paragraph 78 or 79, wherein the buffer is a Tris buffer.

81. The composition of paragraph 80, wherein the Tris buffer comprises tromethamine and Tris hydrochloride (HCl).

82. The composition of paragraph 81, wherein the tromethamine is at a concentration of about 0.1-0.3 mg/mL or about 0.01-0.15 mg/mL.

83. The composition of paragraphs 81 or 82, wherein the Tris HCl is at a concentration of about 1.25-1.40 mg/mL or about 0.5-0.65 mg/mL.

84. The composition of any one of paragraphs 78 to 83, wherein the stabilizing agent is sucrose.

85. The composition of paragraph 84, wherein the sucrose is at a concentration of about 95-110 mg/mL or about 35-50 mg/mL.

86. A composition according to any one of paragraphs 78 to 85, wherein the salt diluent for reconstitution is sodium chloride.

87. The composition of paragraph 86, wherein the sodium chloride is at a concentration of about 5 to 15 mg/mL.

88. A composition according to any one of paragraphs 62 to 87, which is liquid or lyophilized.

89. The composition of paragraph 62, comprising an RNA molecule at a concentration of about 0.01-0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a neutral lipid at a concentration of about 0.1-0.25 mg/mL, and a steroid or steroid analog at a concentration of about 0.3-0.45 mg/mL, and further comprising a Tris buffer comprising tromethamine at a concentration of about 0.1-0.3 mg/mL and Tris hydrochloride (HCl) at a concentration of about 1.25-1.40 mg/mL, and sucrose at a concentration of about 95-110 mg/mL, wherein the composition is a liquid composition.

90. The composition of paragraph 62, comprising an RNA molecule at a concentration of about 0.01-0.09 mg/mL formulated in a lipid nanoparticle (LNP) comprising a cationic lipid at a concentration of about 0.8-0.95 mg/mL, a PEGylated lipid at a concentration of about 0.05-0.15 mg/mL, a neutral lipid at a concentration of about 0.1-0.25 mg/mL, and a steroid or steroid analog at a concentration of about 0.3-0.45 mg/mL, and further comprising a Tris buffer comprising tromethamine at a concentration of about 0.01-0.15 mg/mL and Tris hydrochloride (HCl) at a concentration of about 0.5-0.65 mg/mL, and sucrose at a concentration of about 35-50 mg/mL.

91. The composition of paragraph 90, which is reconstituted with sodium chloride at a concentration of about 5-15 mg/mL.

92. The composition of paragraph 91, which is reconstituted with about 0.6 to 0.75 mL of sodium chloride.

93. The composition of paragraph 62, further comprising about 5-15 mM Tris buffer, 200-400 mM sucrose at a pH of about 7.0-8.0, and optionally a 0.9% sodium chloride diluent for reconstitution.

94. A method for inducing an immune response against VZV in a subject, comprising administering to the subject an effective amount of an RNA molecule and/or composition described in any one of paragraphs 1 to 93.

95. A method for preventing, treating or ameliorating an infection, disease or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule and/or composition described in any one of paragraphs 1 to 93.

96. The method of paragraph 95, wherein the infection, disease or condition is associated with VZV.

97. The method of paragraph 95 or 96, wherein the infection, disease or condition is herpes zoster (shingles).

98. The method of paragraph 95 or 96, wherein the infection, disease or condition is post-herpetic neuralgia.

99. Use of an RNA molecule and/or composition described in any one of paragraphs 1 to 93 in the manufacture of a medicament for use in inducing an immune response against VZV in a subject.

100. Use of an RNA molecule or composition described in any one of paragraphs 1 to 93 in the manufacture of a medicament for use in the prevention, treatment or amelioration of an infection, disease or condition in a subject.

101. The use of paragraph 100, wherein the infection, disease or condition is associated with VZV.

102. The use of paragraph 100 or 101, wherein the infection, disease or condition is herpes zoster (shingles).

103. The use of paragraph 100 or 101, wherein the infection, disease or condition is post-herpetic neuralgia.

104. The method or use of any one of paragraphs 94 to 103, wherein the subject is less than about 1 year old, about 1 year or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

105. The method or use of any one of paragraphs 94 to 104, wherein the subject is about 50 years of age or older.

106. The method or use of any one of paragraphs 94 to 105, wherein the subject is immunocompetent.

107. The method or use of any one of paragraphs 94 to 105, wherein the subject is immunocompromised.

108. The method or use of any one of paragraphs 94 to 107, wherein the RNA molecule and/or composition is administered as a vaccine.

109. The method or use of any one of paragraphs 94 to 108, wherein the RNA molecule and/or composition is administered by intradermal or intramuscular injection.

110. The method or use of any one of paragraphs 94 to 109, wherein the subject is administered a single dose, two doses, three doses or more doses of the RNA molecule and/or composition.

111. The method or use of any one of paragraphs 94 to 110, wherein the subject is administered a single dose of the RNA molecule and/or composition.

112. The method or use of any one of paragraphs 94 to 110, wherein the subject is administered two doses of the RNA molecule and/or composition.

113. The method or use of any one of paragraphs 94 to 110, wherein the subject is administered two doses of the RNA molecule and/or composition on day 0 and about two months later.

114. The method or use of any one of paragraphs 94 to 110, wherein the subject is administered two doses of the RNA molecule and/or composition on day 0 and about 6 months later.

115. The method or use of any one of paragraphs 94 to 114, wherein the subject is administered at least one booster dose of the RNA molecule and/or composition.

116. The method or use of any one of paragraphs 94 to 115, wherein the subject is administered a dose of at least about 15 μg, at least about 30 μg, at least about 60 μg, at least about 90 μg, at least about 100 μg or more of the RNA molecule and/or composition per administration.

117. The method or use of any one of paragraphs 94 to 116, wherein the subject is administered an injection having a volume of about 0.25 to 1 mL, including but not limited to about 0.25, 0.5, 1 mL.

All of the methods disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present disclosure have been described with respect to certain specific embodiments, it will be apparent to those skilled in the art that variations may be applied to the methods and steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. More particularly, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

The contents of all references cited in this application (including literature references, issued patents, patent application publications, and GENBANK® Accession Numbers referenced throughout this application), to the extent that they provide exemplary procedural or other details supplementing the details set forth herein, are specifically and expressly incorporated herein by reference. In the event that a definition of a term in a document incorporated herein by reference conflicts with a definition used herein, the definition used herein shall control.

Claims (32)

A composition for inducing an immune response against varicella zoster virus (VZV) in a subject, comprising an RNA molecule comprising at least one open reading frame encoding a VZV glycoprotein E (gE) polypeptide. A composition for preventing, treating, or ameliorating an infection, disease, or condition associated with Varicella-Zoster Virus (VZV) in a subject, comprising an RNA molecule comprising at least one open reading frame encoding a VZV glycoprotein E (gE) polypeptide. Use of an RNA molecule comprising at least one open reading frame encoding a varicella zoster virus (VZV) glycoprotein E (gE) polypeptide in the manufacture of a medicament for use in the prevention, treatment or amelioration of an infection, disease or condition associated with VZV in a subject. The composition of claim 2, wherein the infection, disease or condition is herpes zoster (shingles) or post-herpetic neuralgia. The use according to claim 3, wherein the infection, disease or condition is herpes zoster (shingles) or post-herpetic neuralgia. Use of an RNA molecule comprising at least one open reading frame encoding a varicella zoster virus (VZV) glycoprotein E (gE) polypeptide in the manufacture of a medicament for use in inducing an immune response to VZV in a subject. The composition or use according to any one of claims 1 to 6, wherein the VZV polypeptide is full length, truncated, fragment or variant thereof. The composition or use of any one of claims 1 to 7, wherein the VZV polypeptide comprises at least one mutation. The composition or use according to any one of claims 1 to 8, wherein the VZV polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the amino acid sequences selected from SEQ ID NOs: 1 to 11. The composition or use according to any one of claims 1 to 9, wherein the open reading frame is transcribed from a nucleic acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% identity to any one of the sequences selected from SEQ ID NOs: 12 to 145. The composition or use of any one of claims 1 to 10, 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 selected from SEQ ID NOs: 146 to 279. The composition or use according to any one of claims 1 to 11, wherein the open reading frame comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 146 to 279. The composition or use of any one of claims 1 to 12, further comprising a 5' untranslated region (5'UTR). The composition or use of claim 13, wherein the 5'UTR comprises a sequence selected from any one of SEQ ID NOs: 281, 312, or 313. The composition or use of any one of claims 1 to 14, further comprising a 3' untranslated region (3'UTR). 16. The composition or use of claim 15, wherein the 3'UTR comprises a sequence selected from any one of SEQ ID NOs: 284, 314, or 317. The composition or use of any one of claims 1 to 16, further comprising a 5' cap portion and/or a 3' poly A tail. The composition or use of claim 17, wherein the polyA tail comprises a sequence selected from any one of SEQ ID NOs: 287 or 315. The composition or use of any one of claims 1 to 18, wherein the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, about 50%-75%, or about 55%-70%. 20. The composition or use of any one of claims 1 to 19, wherein the encoded VZV polypeptide is localized in the cell membrane, localized in the Golgi, and/or secreted. The composition or use of any one of claims 1 to 20, wherein the RNA comprises at least one modified nucleotide. A composition or use according to any one of claims 1 to 21, wherein each uridine is replaced by N1-methylpseudouridine (Ψ). The composition or use according to any one of claims 1 to 22, wherein the RNA is mRNA. A composition comprising an RNA molecule according to any one of claims 1, 2, 4 or 7 to 23, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). 25. The composition of claim 24, wherein the lipid nanoparticles comprise at least one of a cationic lipid, a PEGylated lipid, a neutral lipid, and a steroid or steroid analog. The composition according to claim 25, wherein the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). PEGylated lipids include PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol)2000)carbamyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), and glycol lipids including PEG-2000-DMG, PEG-modified diacylglycerols (PEG-DAG), e.g., 1 The composition according to claim 25 or 26, which is selected from the group consisting of -(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), and PEG dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate. Neutral lipids include 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine (DOPE). The composition according to any one of claims 25 to 27, wherein the phosphatidylethanolamine is 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-dielideyl-sn-glycero-3-phosphoethanolamine (trans DOPE). The composition of any one of claims 25 to 28, wherein the steroid or steroid analog is cholesterol. 23. The composition or use of any one of claims 1 to 22, wherein the subject is less than about 1 year old, about 1 year or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. The composition of any one of claims 1, 2, 4, or 7 to 30, wherein the RNA molecule or composition is administered as a vaccine. The composition or use of any one of claims 1 to 31, wherein the subject is administered a single dose, two doses, three doses or more doses of the RNA molecule or composition, and optionally a booster dose.

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