CN118574637A - RNA molecules - Google Patents

Abstract

The present disclosure relates to RNA molecules encoding varicella zoster virus (VZV). The present disclosure further relates to compositions comprising RNA molecules formulated in lipid nanoparticles (RNA-LNPs). The present disclosure further relates to the use of RNA molecules, RNA-LNPs, and compositions for treating or preventing herpes zoster (shingles).

Description

RNA molecules

Background Art

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 recurs as herpes zoster or shingles. Two vaccines have been developed and licensed in various countries to prevent herpes zoster. The first vaccine is (Merck & Co., Inc., Kenilworth, NJ, USA), a live attenuated VZV vaccine. Approved by US FDA in 2006 However, as of November 2020, No longer sold in the U.S. The second vaccine is (GlaxoSmithKline, Rockville, MD, USA), a VZV gE subunit protein vaccine with AS01 _B as adjuvant. Approved by US FDA in 2017

In the United States, approximately one million cases of shingles occur each year, and approximately 30% of all people infected with chickenpox will 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.

Summary of the invention

The present disclosure provides immunogenic compositions and methods for treating a subject, the methods comprising administering an RNA molecule (e.g., an immunogenic RNA polynucleotide) encoding an amino acid sequence (e.g., an immunogenic antigen) comprising 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. RNA polynucleotides encoding immunogenic antigens are administered to provide antigens for inducing (e.g., stimulating, priming, and/or amplifying) an immune response (e.g., antibodies and/or immune effector cells) (after expression of the polynucleotide by appropriate target cells). In one aspect, the immune response to be induced according to the present disclosure is a B cell-mediated immune response, such as an antibody-mediated immune response. Additionally or alternatively, the immune response to be induced according to the present disclosure may be a T cell-mediated immune response. In one aspect, the immune response is an anti-VZV immune response.

The immunogenic compositions described herein include RNA molecules, which include RNA (as active ingredients) that can be translated into proteins in recipient cells. In addition to the wild-type or codon-optimized sequences encoding antigenic sequences, RNA molecules may contain one or more structural components (5' end caps, 5'UTR, 3'UTR, polyadenylic acid tails) optimized for the maximum effectiveness of RNA for stability and translation efficiency. In one aspect, RNA molecules contain all these components. In some aspects, each uridine of RNA molecules is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA). RNA molecules described herein can be deployed together with lipids and/or proteins, encapsulated in lipids and/or proteins, or compounded with lipids and/or proteins to produce RNA-particles (e.g., lipid nanoparticles (LNPs)) for administration. In one aspect, RNA molecules described herein are deployed together with lipids, encapsulated in lipids, or compounded with lipids to produce RNA-lipid nanoparticles (e.g., RNA-LNPs) for administration. In one aspect, RNA molecules described herein are deployed together with proteins, encapsulated in proteins, or compounded with proteins for administration. In one aspect, the RNA molecules described herein are formulated with lipids and proteins, encapsulated in lipids and proteins, complexed with lipids and proteins for administration. If a combination of different RNA molecules is used, the RNA molecules can be formulated together with lipids and/or proteins or separately formulated with lipids and/or proteins to produce RNA-particles for administration.

The present disclosure provides RNA molecules and RNA-LNPs, which include at least one open reading frame (ORF) encoding a VZV antigen. In some aspects, the VZV antigen is a VZV polypeptide. In some aspects, the VZV polypeptide is a VZV glycoprotein. In some aspects, the VZV glycoprotein is VZV gK, gN, gC, gB, gH, gM, gL, gI or gE. In some aspects, the VZV glycoprotein is VZVgE. In some aspects, the VZV polypeptide is a full-length VZV polypeptide, a truncated VZV polypeptide, a fragment of a VZV polypeptide or a variant of a VZV polypeptide. In some aspects, 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 aspects, the VZV polypeptide has, has at least, or has at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to any of the amino acid sequences of Table 1, e.g., to any of SEQ ID NOs: 1 to 11. In some aspects, the VZV polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 11. In some aspects, the VZV polypeptide consists of any of the amino acid sequences of Table 1, e.g., consists of any of SEQ ID NOs: 1 to 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 aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having, having at least, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or more identity to any one of the nucleic acid sequences of Table 2, e.g., to any one of SEQ ID NOs: 12 to 145. In some aspects, the RNA molecule is transcribed from a nucleic acid sequence selected from SEQ ID NOs: 12 to 145. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any one of the nucleic acid sequences of Table 2, e.g., consisting of any one of SEQ ID NOs: 12 to 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 aspects, the RNA molecule comprises a nucleic acid sequence having, having at least, or having at most 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher identity with any one of the nucleic acid sequences of Table 3, e.g., any one of SEQ ID NOs: 146 to 279. In some aspects, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 146 to 279. In some aspects, the RNA molecule comprises a nucleic acid sequence consisting of any one of the nucleic acid sequences of Table 3, e.g., any one of SEQ ID NOs: 146 to 279. In some aspects, each uridine of any one of SEQ ID NOs: 146 to 279 is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure further provides RNA molecules and RNA-LNPs, which include a 5' untranslated region (5'-UTR) and/or a 3' untranslated region (3'-UTR). In some aspects, the RNA molecule includes a 5' untranslated region (5'-UTR). In some aspects, the 5'UTR includes a sequence selected from any one of SEQ ID NO: 281 (SEQ ID NO: 280-DNA; SEQ ID NO: 282-RNA with Ψ) and 312 to 313. In some aspects, the 5'UTR includes a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher identity to any one of SEQ ID NO: 281 and 312 to 313. In some aspects, the 5'UTR includes a sequence selected from any one of SEQ ID NO: 281 and 312 to 313. In some aspects, the 5'UTR comprises a sequence consisting of any one of SEQ ID NOs: 281 and 312-313.

In some aspects, the RNA molecules and RNA-LNPs include a 3' untranslated region (3'-UTR). In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 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 aspects, the 3'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or higher identity with any one of SEQ ID NO: 284, 314 and 317. In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 284, 314 and 317. In some aspects, the 3'UTR comprises a sequence consisting of any one of SEQ ID NO: 284, 314 and 317.

The present disclosure further provides RNA molecules and RNA-LNPs, which include a 5' end cap portion. In some aspects, the 5' end cap portion is (3'OMe)-m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG. The present disclosure further provides RNA molecules and RNA-LNPs, which include a 3' poly(A) tail. In some aspects, the poly(A) tail comprises a sequence selected from any one of SEQ ID NO: 287 (SEQ ID NO: 286-DNA; SEQ ID NO: 288-RNA with Ψ) and 315 (SEQ ID NO: 316-RNA with Ψ). In some aspects, the poly(A) tail comprises a sequence selected from any one of SEQ ID NO: 287 and 315 +/- 1 adenosine (A) or +/- 2 adenosines (A).

In some aspects, the RNA molecule includes a 5'UTR and a 3'UTR. In some aspects, the RNA molecule includes a 5' end cap, a 5'UTR and a 3'UTR. In some aspects, the RNA molecule includes a 5' end cap, a 5'UTR, a 3'UTR and a poly(A) tail. In some aspects, the RNA molecule includes a 5'UTR, a 3'UTR and a poly(A) tail. In some aspects, each uridine of any one of the 5'UTR, the 3'UTR and the poly(A) tail is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure provides RNA molecules as described in Table 5. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315 (gE WT). In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315 (gE WT CO1). In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315 (gE WT CO2). In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315 (ms3CO1). In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315 (ms3CO2). In some aspects, 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 poly(A) tail (ms4 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms4 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms5 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms5 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms5 CO2 v2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms6 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms6 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms8 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms9 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms9 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms10 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms10 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms10 CO3) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms11 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms11 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms12 CO1) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail (ms12 CO2) of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, 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 to 238, a 3'UTR of SEQ ID NOs: 284 or 317, and/or a poly(A) tail of SEQ ID NOs: 287 or 315. In some aspects, the RNA molecule comprises a 5'UTR of SEQ ID NO: 281 or 312, a VZVORF of SEQ ID NO: 239, a 3'UTR of SEQ ID NOs: 284 or 317, and/or a poly(A) tail of SEQ ID NOs: 287 or 315. In some aspects, the RNA molecule comprises a 5'UTR of SEQ ID NO: 281 or 312, a VZVORF of any one of SEQ ID NOs: 240 to 254, a 3'UTR of SEQ ID NOs: 284 or 317, and/or a poly(A) tail of SEQ ID NOs: 287 or 315. In some aspects, the RNA molecule comprises a 5'UTR of SEQ ID NO: 281 or 312, a VZVORF of any one of SEQ ID NO: 255 to 267, a 3'UTR of SEQ ID NO: 284 or 317, and/or a poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, the RNA molecule comprises a 5'UTR of SEQ ID NO: 281 or 312, a VZVORF of any one of SEQ ID NO: 268 to 279, a 3'UTR of SEQ ID NO: 284 or 317, and/or a poly(A) tail of SEQ ID NO: 287 or 315. In some aspects, the VZV ORF further comprises a stop codon as described herein. In some aspects, the poly(A) tail length may contain +1/-1A or +2/-2A. In some aspects, each uridine of the RNA molecule is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA).

The present disclosure further provides RNA molecules comprising at least one open reading frame produced by codon-optimized DNA. In some aspects, 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% to 75%, or about 55% to 70%. In some aspects, the G/C content is about 58%, about 66% or about 62%. The present disclosure further provides RNA molecules encoding VZV polypeptides, which are located in the cell membrane, in the Golgi apparatus and/or anchored in the membrane and secreted. The present disclosure further provides RNA molecules comprising stabilized RNA. The present disclosure further provides RNA molecules comprising RNA having at least one modified nucleotide (e.g., modified RNA; modRNA). In some aspects, the modified nucleotide is pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine. In some aspects, the modified nucleotide is N1-methyl pseudouridine (Ψ).

The present disclosure further provides RNA molecules that are messenger RNA (mRNA) or self-replicating RNA. In some aspects, the RNA is mRNA.

The disclosure further provides immunogenic compositions comprising RNA molecules described herein. In such immunogenic compositions, the RNA molecules can be formulated in lipid nanoparticles (LNPs), encapsulated in LNPs, compounded with LNPs, bound to LNPs, or adsorbed on LNPs (e.g., VZV RNA-LNPs). In some aspects, the lipid nanoparticles include 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 aspects, the lipid nanoparticles include a cationic lipid. In some aspects, the cationic lipid is (4-hydroxybutyl) azanediyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).

In some aspects, the lipid nanoparticles include polymer-conjugated lipids. In some aspects, the lipid nanoparticles include pegylated lipids, also known as PEG-lipids. In some aspects, the PEGylated lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide, a glycol lipid (including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxypropane-3-amine (PEG-c-DMA) and PEG-2000-DMG), a PEGylated diacylglycerol (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyloxypropane-3-amine ... In some aspects, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-di-tetradecanoyl)acetamide (ALC-0159).

In some aspects, the lipid nanoparticles include at least one structural lipid, such as a neutral lipid. In some aspects, the neutral lipid is selected from 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. 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE) and/or 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In some aspects, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

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

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

In some aspects, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, wherein the concentration of the RNA molecule/polynucleotide is at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, and the RNA molecule/polynucleotide is encapsulated in an LNP having a lipid composition 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL. In some aspects, the liquid composition further comprises a buffer composition comprising a first buffer at a concentration of about 0.15 to 0.3 mg/mL, a second buffer at a concentration of about 1.25 to 1.4 mg/mL, and a stabilizer at a concentration of about 95 to 110 mg/mL.

In a particular aspect, the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, at a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, encapsulated in an LNP having a lipid composition comprising: about 0.8 to 0.95 mg/mL, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (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. In some aspects, the liquid composition further comprises a Tris buffer composition containing tromethamine at a concentration of about 0.1 to 0.3 mg/mL and Tris hydrochloric acid (HCl) at a concentration of about 1.25 to 1.4 mg/mL, and sucrose at a concentration of about 95 to 110 mg/mL.

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

In some aspects, the RNA-LNP immunogenic composition is a lyophilized (reconstituted) RNA-LNP composition comprising an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, wherein the concentration of the RNA molecule/polynucleotide is at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, and the RNA molecule/polynucleotide is encapsulated in an LNP having a lipid composition 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL. In some aspects, the lyophilized composition further comprises a first buffer at a concentration of about 0.01 and 0.15 mg/mL, a second buffer at a concentration of about 0.5 and 0.65 mg/mL, a stabilizer at a concentration of about 35 to 50 mg/mL, and a salt diluent for reconstitution at a concentration of about 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in about 0.6 to 0.75 mL of the salt diluent. The concentration in the lyophilized RNA-LNP composition is determined after reconstitution.

In a particular aspect, the lyophilized (reconstituted) RNA-LNP composition comprises an RNA polynucleotide encoding a VZV polypeptide as disclosed herein, at a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, encapsulated in LNPs having a lipid composition of about 0. 8 to 0.95 mg/mL of ALC-0315, about 0.05 to 0.15 mg/mL of ALC-0159, about 0.1 to 0.25 mg/mL of DSPC, and about 0.3 to 0.45 mg/mL of cholesterol; and the lyophilized (reconstituted) RNA-LNP composition further comprises a Tris buffer composition containing tromethamine at a concentration of about 0.01 and 0.15 mg/mL and Tris HCl at a concentration of about 0.5 and 0.65 mg/mL, sucrose at a concentration of about 35 to 50 mg/mL, and a sodium chloride (NaCl) diluent for reconstitution at a concentration of about 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in about 0.6 to 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, at most, exactly, or any between the following per administration: 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more 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., 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 can be administered twice, at day 0 and about 6 months later. The present disclosure further provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times or more. In some aspects, periodic booster immunizations at intervals of 1 to 5 years may be required to maintain the protective level of antibodies. The present disclosure further provides the administration of at least one booster dose.

The present disclosure provides a method of inducing an immune response against VZV in a subject, comprising administering to the subject an effective amount of the RNA molecules, RNA-LNPs and/or immunogenic compositions described herein. The present disclosure further provides the use of the RNA molecules, RNA-LNPs and/or immunogenic compositions described herein for preparing a medicament for inducing an immune response against VZV in a subject.

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

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

The present disclosure provides a method for preventing, treating or improving an infection, disease or condition of a subject, comprising administering to a subject an effective amount of RNA molecules, RNA-LNPs and/or immunogenic compositions described herein. The present disclosure further provides the use of RNA molecules, RNA-LNPs and/or immunogenic compositions described herein for the preparation of a medicament for preventing, treating or improving an infection, disease or condition of a subject. In some aspects, the infection, disease or condition is associated with VZV. In some aspects, the infection, disease or condition is herpes zoster (herpes zoster) (shingles). In some aspects, the infection, disease or condition is postherpetic neuralgia.

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

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

In some aspects, the subject is, is at least, or is at most less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. In some aspects, the subject is about 50 years old or older.

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

The present disclosure provides methods or uses described herein, wherein the RNA molecules, RNA-LNPs and/or immunogenic compositions are administered as vaccines.

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 in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating certain aspects of the present disclosure, are given by way of illustration only, because 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 schematically shows wild-type (WT) varicella zoster virus (VZV) gE protein (gE WT) and variant VZVgE protein, wherein SP refers to a signal peptide sequence, an extracellular domain refers to a peptide sequence corresponding to a portion of the protein extending into the extracellular space, TM refers to a transmembrane peptide sequence corresponding to a portion of the protein spanning the cell membrane, and CT refers to a cytoplasmic tail peptide sequence corresponding to a portion of the protein extending into the cytoplasm. Variant VZV gE proteins with cytoplasmic tail modifications are referred to as ms4, ms5, ms8, ms9, ms10, ms11, and ms12. Secretory variant VZV gE proteins with TM modifications are referred to as ms3 and ms6. VZV gE RNA constructs encoding VZV gE proteins are generated from codon-optimized (CO) DNA, wherein CO1 indicates a CO construct with about 58% G/C content, CO2 indicates a CO construct with about 66% G/C content, and CO3 indicates a CO construct with about 62% G/C content.

Figure 2 shows VZV gE expression in 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; each with two constructs CO1 and CO2). Cells were imaged for VZV gE expression, and the percentage of VZV gE ⁺ transfected Vero cells for each VZV RNA construct is shown.

Figure 3 shows VZV gE expression in Vero cells transfected with 10 ng, 25 ng or 50 ng of VZV RNA constructs (secreted) with modifications in the transmembrane (TM) (ms3 and ms6; two constructs CO1 and CO2, respectively). Cells were imaged for VZV gE expression, and the percentage of VZV gE ⁺ transfected Vero cells is shown for each VZV RNA construct.

Figure 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; two constructs for CO1 and CO2 each).

Figure 5 shows the mean fluorescence intensity (MFI) of Vero cells transfected with 10 ng, 25 ng or 50 ng of VZV RNA constructs (secretory) with modifications in the transmembrane (TM) (ms3 and ms6; two constructs CO1 and CO2, respectively).

FIG. 6 shows a 63-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 50 ng of gE WT VZV RNA construct and variant VZV gE RNA constructs with modified cytoplasmic tails (ms4, ms5, ms8).

FIG. 7 shows a 63-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 50 ng of variant VZV gE RNA constructs with modified cytoplasmic tails (ms9, ms11, and ms12).

FIG. 8 shows a 63-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 50 ng of a variant VZV gE RNA construct with a modified cytoplasmic tail (ms10).

FIG. 9 shows a 63-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 50 ng of variant VZV gE RNA constructs (ms3 and ms6) with TM modifications (secretory).

FIG. 10 shows a 10-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 25 ng of gE WT VZV RNA construct and variant VZVgE RNAs with modified cytoplasmic tails (ms4, ms5, ms8).

FIG. 11 shows a 10-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 25 ng of variant VZV gE RNA constructs with modified cytoplasmic tails (ms9, ms11, ms12).

FIG. 12 shows a 10-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 25 ng of a variant VZV gE RNA construct with a modified cytoplasmic tail (ms10).

FIG. 13 shows a 10-fold magnification of subcellular localization of VZV gE in Vero cells transfected with 25 ng of variant VZV gE RNA constructs with TM modifications (ms3 and ms6) (secretory).

Figure 14 shows a titration curve generated by plotting the amount of input RNA relative to the percentage of human embryonic kidney (HEK) 293T cells expressing VZV gE, wherein the titration curve was used to determine the _EC50 of VZV RNA-LNP vaccines formulated with the following: VZV gE WT RNA constructs (gE WT CO1 and gE WT CO2), variant VZV gE RNA constructs with cytoplasmic tail modifications (ms4 CO1), and variant VZV gE RNA constructs with transmembrane (TM) modifications (ms3CO1) (secreted).

Figure 15 shows a titration curve generated by plotting the amount of input RNA relative to the percentage of human embryonic kidney (HEK) 293T cells expressing VZV gE, wherein the titration curve was used to determine the _EC50 of VZV RNA-LNP vaccines formulated with the following: VZV gE WT RNA constructs (gE WT CO1 and gE WT CO2), variant VZV gE RNA constructs with cytoplasmic tail modifications (ms5 CO1), and variant VZV gE RNA constructs with TM modifications (ms6 CO2) (secreted).

Figure 16 shows serum IgG levels at day 28 post-immunization in mice immunized on day 0 with priming doses of: 1 μg, 2.5 μg, or 5 μg 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with gE_WT CO1RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with ms3 CO1 RNA construct with TM modification (secretory); 0.5 μg of VZV RNA-LNP vaccine formulated with ms4 CO1 RNA construct with cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine (gE_WT CO1 LYO) formulated with gE_WT CO1RNA construct.

Figure 17 shows serum IgG levels at day 34 post-immunization in mice immunized with a prime dose of the following on day 0 and a booster dose of the corresponding ones on day 28: 1 μg, 2.5 μg, or 5 μg 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with gE_WT CO1 RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with ms3 CO1 RNA construct with TM modification (secretory); 0.5 μg of VZV RNA-LNP vaccine formulated with ms4 CO1 RNA construct with cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine (gE_WT CO1 LYO) formulated with gE_WT CO1 RNA construct.

Figure 18 shows serum IgG levels at day 42 post-immunization in mice immunized on day 0 with a prime dose of each of the following and on day 28 with a booster dose of the corresponding: 1 μg, 2.5 μg, or 5 μg 0.5 μg or 1 μg of VZV RNA-LNP vaccine formulated with gE_WT CO1 RNA construct; 0.5 μg of VZV RNA-LNP vaccine formulated with ms3 CO1 RNA construct with TM modification (secretory); 0.5 μg of VZV RNA-LNP vaccine formulated with ms4 CO1 RNA construct with cytoplasmic tail modification; 0.5 μg of VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct; or 0.5 μg or 1 μg of lyophilized VZV RNA-LNP vaccine (gE_WT CO1 LYO) formulated with gE_WT CO1 RNA construct.

Figure 19 shows the comparison of serum IgG levels in mice at day 28 (priming dose only), day 34 (six days after boosting dose) and day 42 (two weeks after boosting dose) after immunization. The subjects were immunized with either (1 μg) or VZV RNA-LNP vaccine (0.5 μg) and with a booster dose of the respective one on day 28.

Figure 20 shows the percentage of CD4 ⁺ IFN-γ ⁺ stained T cells after in vitro stimulation of splenocytes harvested from mice on day 34 after immunization. The patients were immunized with either 1 μg, 2.5 μg, or 5 μg of VZV RNA-LNP vaccine (0.5 μg and/or 1 μg) and with a booster dose of the respective one on day 28.

Figure 21 shows the percentage of CD8 ⁺ IFN-γ ⁺ stained T cells after in vitro stimulation of splenocytes harvested from mice on day 34 after immunization. The patients were immunized with either 1 μg, 2.5 μg, or 5 μg of VZV RNA-LNP vaccine (0.5 μg and/or 1 μg) and with a booster dose of the respective one on day 28.

FIG. 22 shows serum IgG levels at day 35 after subcutaneous vaccination in mice vaccinated subcutaneously on day 0 with 1350 pfu of live attenuated varicella (LAV).

FIG. 23 shows serum IgG levels at day 63 post-vaccination (1 month after the 1st dose) in mice vaccinated with LAV vaccine on day 0 and immunized on day 35 with one dose of: (5μg, 2.5μg or 1μg); VZV RNA-LNP vaccine formulated with gE_WT CO2RNA construct (1μg or 0.5μg); VZV RNA-LNP vaccine formulated with ms5 CO1 RNA construct with cytoplasmic tail modification (1μg or 0.5μg); or VZV RNA-LNP vaccine formulated with ms6 CO2 RNA construct (secretory) with TM modification (1μg or 0.5μg).

FIG. 24 shows serum IgG levels at day 76 post-vaccination (13 days after 2nd dose/boost) in mice vaccinated with LAV vaccine on day 0 and immunized on days 35 and 63 with a dose of: (5μg, 2.5μg or 1μg); VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (1μg or 0.5μg); VZV RNA-LNP vaccine formulated with ms5 CO1 RNA construct with cytoplasmic tail modification (1μg or 0.5μg); or VZV RNA-LNP vaccine formulated with ms6 CO2 RNA construct (secretory) with TM modification (1μg or 0.5μg).

Figure 25 shows serum IgG levels at day 63 post-vaccination (1 month after the 1st dose) in mice vaccinated with LAV vaccine on day 0 and immunized with one dose of the following on day 35: VZV RNA-LNP vaccine formulated with gE_WTCO2 RNA construct (1 μg or 0.5 μg) or lyophilized VZV RNA-LNP vaccine formulated with gE_WTCO2 RNA construct (gE_WTCO2lyo) (1 μg or 0.5 μg); and serum IgG levels at day 76 post-vaccination (13 days after the 2nd dose/boost) in mice vaccinated with LAV vaccine on day 0 and immunized with one dose of the following on days 35 and 63: VZV RNA-LNP vaccine formulated with gE_WTCO2 (1 μg or 0.5 μg) or lyophilized VZV RNA-LNP vaccine formulated with gE_WTCO2RNA construct (gE_WTCO2lyo) (1 μg or 0.5 μg). CO2 lyo)(1μg or 0.5μg).

FIG. 26A-D show vaccine-induced VZV gE-specific T and B cell responses in splenocytes collected on day 48 (13 days after the first dose) from LAV-experienced mice immunized on day 35 with one dose of: (5μg or 1μg); VZV RNA-LNP vaccine formulated with gE_WT CO2 RNA construct (1μg or 0.5μg); VZV RNA-LNP vaccine formulated with ms5 CO1 RNA construct with cytoplasmic tail modification (1μg); VZV RNA-LNP vaccine formulated with ms6 CO2 RNA construct with TM modification (1μg); or freeze-dried VZV RNA-LNP vaccine (gE_WT CO2 Lyo) formulated with gE_WT CO2 (1μg). Figure 26A shows the number of VZV gE-specific cells secreting IFN-γ measured by ELISpot, and the results are expressed as spot-forming cells (SFC) per million cells. Figure 26B shows cells expressing IFN-γ in CD4 ⁺ T cells measured by ICS analysis, which is expressed as the percentage of IFN-γ ⁺ cells in CD4 ⁺ T cells. Figure 26C shows IFN-γ expressing cells within CD8 ⁺ T cells measured by ICS analysis, expressed as the percentage of IFN-γ ⁺ cells within CD8+ T cells. Figure 26D shows B cell responses in splenocytes, assessed by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry.

FIG. 27A-27C show vaccine-induced VZV gE-specific T and B cell responses in splenocytes collected on day 76 (13 days after the 2nd dose/boost) in LAV-experienced mice immunized on days 35 and 63 with a dose of: (5μg or 1μg); VZV RNA-LNP vaccine formulated with gE_WT CO2RNA construct (1μg or 0.5μg); VZV RNA-LNP vaccine formulated with ms5 CO1 RNA construct with cytoplasmic tail modification (1μg); VZV RNA-LNP vaccine formulated with ms6 CO2RNA construct with TM modification (1μg); or freeze-dried VZV RNA-LNP vaccine (gE_WT CO2 Lyo) formulated with gE_WT CO2 (1μg). Figure 27A shows cells expressing IFN-γ in CD4 ⁺ T cells measured by ICS analysis, which is expressed as the percentage of IFN-γ ⁺ cells in CD4 ⁺ T cells. Figure 27B shows cells expressing IFN-γ in CD8 ⁺ T cells measured by ICS analysis, which is expressed as the percentage of IFN-γ ⁺ cells in CD8+T cells. FIG. 27C shows B cell responses in splenocytes, assessed by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides an RNA molecule (e.g., an RNA polynucleotide) comprising at least one open reading frame (ORF) encoding a varicella zoster virus (VZV) antigen. In some aspects, the VZV antigen is a VZV polypeptide. In some aspects, the VZV polypeptide is a VZV gE polypeptide. In some aspects, the VZV polypeptide comprises the amino acid sequence of Table 1. In some aspects, the RNA molecule comprises an ORF transcribed from at least one DNA nucleic acid sequence of Table 2. In some aspects, the RNA molecule comprises an ORF containing an RNA nucleic acid sequence of Table 3. In some aspects, the RNA molecule comprises at least one of a 5' end cap, a 5'UTR, a 3'UTR, and a polyadenylic acid tail. The present disclosure provides an RNA molecule comprising a modified nucleotide (e.g., a modified RNA; modRNA). The present disclosure provides an immunogenic composition comprising any one of the RNA molecules encoding the VZV polypeptides described herein, the RNA molecule being complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forming lipid nanoparticles (RNA-LNP). The disclosure further provides an immunogenic composition comprising any of the RNA molecules containing at least one RNA nucleic acid described herein, the RNA molecules being complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forming RNA-LNPs. The disclosure further provides a method of preventing, treating, or ameliorating an infection, disease, or condition (e.g., herpes zoster or shingles) in a subject by administering to the subject an effective amount of the RNA molecules, RNA-LNPs, or immunogenic compositions described herein. The disclosure further provides the use of the RNA molecules, RNA-LNPs, and/or immunogenic compositions described herein as vaccines.

I. Examples of Definitions

Throughout this application, the term "about" is used according to its ordinary and customary meaning in the arts of cell and molecular biology to indicate a ±10% deviation from the value or values in connection with which it is used.

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

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

The phrase "and/or" means "and" or "or". For illustration, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, "and/or" acts as an inclusive or operation.

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

Compositions and methods, when used, may "comprise," "consist essentially of," or "consist of" any of the ingredients or steps disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and should be understood to imply the inclusion of a stated step or element, or group of steps or elements, but not the exclusion of the inclusion 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 "comprising" may also be implemented in the context of the term "consisting of" or "consisting essentially of." Compositions and methods that "consist essentially of" any of the disclosed ingredients or steps limit the scope of the claim to the specified substances or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. The phrase "consisting of" (and any form of consisting of, such as "consist of" and "consists of") is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or optional, and that no other elements may be present.

References throughout this specification to "one aspect," "an aspect," "particular aspects," "related aspects," "an aspect," "additional aspects," or "another aspect," or combinations thereof, mean that a particular feature, structure, or characteristic described in conjunction with that aspect is included in at least one aspect of the present disclosure. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same aspect. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more aspects.

The terms "inhibiting," "decreasing," or "reducing," or any variation of these terms, include any measurable decrease (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease) or complete inhibition to achieve a desired result. The terms "improving," "enhancing," or "increasing," or any variation of these terms, include any measurable increase (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% increase) to achieve a desired result or 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. A reference, standard, or control can be tested and/or determined substantially simultaneously and/or by the test or determination of interest for the agent, subject, population, sample, or value of interest, and/or can be determined or characterized under conditions or circumstances comparable to the agent, subject, population, sample, or value of interest being assessed.

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

As used herein, "nucleic acid" is a molecule comprising a nucleic acid component and refers to a DNA or RNA molecule. It can be used interchangeably with the term "polynucleotide". Nucleic acid molecules are polymers comprising or consisting of nucleotide monomers, which are covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. Nucleic acids can also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules. Nucleic acids can exist in a variety of forms, such as: isolated segments and recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides (such as one or two chains of an antigen or antibody), or fragments, derivatives, mutant proteins, or variants thereof; polynucleotides sufficient to be used as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting the expression of polynucleotides; mRNA; saRNA; and the complementary sequences of the foregoing described herein. Nucleic acids can encode epitopes to which antibodies can bind.

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

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

The term "polynucleotide" refers to a nucleic acid molecule that may be recombinant or separated from total genomic nucleic acid. The term "polynucleotide" includes oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors (including, for example, plasmids, cosmids, phages, viruses), etc. In certain aspects, a polynucleotide includes a regulatory sequence that is substantially separated from its naturally occurring gene or protein coding sequence. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), an analog thereof, or a combination thereof. Additional coding or non-coding sequences may (but need not) be present in a polynucleotide.

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

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

In this regard, the term "gene" is used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or a portion of a polypeptide may contain a continuous nucleic acid sequence encoding all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid containing variations that have slightly different nucleic acid sequences but still 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 by the nucleic acid sequence. In some aspects, the gene product may be a transcript. In some aspects, the gene product may be a polypeptide. In some aspects, expression of a nucleic acid sequence involves one or more of the following: (1) generation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translation of the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

In general, the term "engineered" refers to aspects that have been manipulated artificially. For example, a polynucleotide is considered "engineered" when two or more sequences that are not linked together in this order in nature are directly linked to each other in an engineered polynucleotide by artificial manipulation and/or when specific residues in a polynucleotide are non-naturally occurring and/or are linked to entities or parts that are not linked in nature by artificial action.

As used herein, the term "DNA" means a nucleic acid molecule comprising nucleotides such as deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate and deoxycytidine monophosphate monomers, which are composed of a sugar portion (deoxyribose), a base portion and a phosphate portion, and are polymerized by a characteristic main chain structure. The main chain structure is usually formed by a phosphodiester bond between the sugar portion (e.g., deoxyribose) of the nucleotide of the first monomer and the phosphate portion of the second adjacent monomer. The specific order of monomers (e.g., the order of bases connected to the sugar/phosphate backbone) is referred to as a DNA sequence. DNA can be single-stranded or double-stranded. In a double-stranded form, the nucleotides of the first chain are usually hybridized with the nucleotides of the second chain, for example, by A/T base pairing and G/C base pairing. DNA may include all or most of the deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide that does not contain a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Without limitation, DNA may encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.

As used herein, the term "RNA" means a nucleic acid molecule comprising nucleotides such as adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate monomers, which are connected to each other along a so-called main chain. The main chain is formed by a phosphodiester bond between the sugar (e.g., ribose) of the first monomer and the phosphate moiety of the second adjacent monomer. RNA can be obtained, for example, by transcription of a DNA sequence in a cell. In eukaryotic cells, transcription is usually performed in the nucleus or mitochondria. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). For example, the processing of immature RNA in eukaryotic organisms includes various post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. Mature messenger RNA is processed and provides a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. Mature mRNA may include a 5' end cap, a 5' UTR, an open reading frame, a 3' UTR and a polyadenylic acid tail sequence. RNA may include all or most of the ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide containing a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. In one aspect, the RNA may be a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known to those skilled in the art, mRNA generally contains a 5' untranslated region (5'UTR), a polypeptide coding region, and a 3' untranslated region (3'UTR). Without limitation, RNA may encompass 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 can be recombinant or has been separated from total genomic nucleic acid. An isolated RNA molecule or protein can exist in a substantially purified form, or can exist in a non-native environment such as a host cell.

"Modified RNA" or "modRNA" refers to an RNA molecule having at least one addition, deletion, substitution and/or change of one or more nucleotides compared to a naturally occurring RNA. Such changes may refer to the addition of non-nucleotide substances to internal RNA nucleotides, or to the 5' and/or 3' ends of the RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as a change in 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 each instance of uridine and/or cytidine in an RNA sequence, or may occur only for selected uridine and/or cytidine nucleotides. Such changes in standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide may be replaced by N1-methyl pseudouridine in an RNA sequence. Other such changed nucleotides are known to those skilled in the art. Such changed RNA molecules are considered to be analogs of naturally occurring RNA. In some aspects, RNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, 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 can be used as a therapeutic modality for treating and/or preventing a variety of conditions in mammals (including humans). The methods described herein include administering RNA described herein to mammals (such as humans). For example, in one aspect, such methods using RNA include RNA vaccines encoding antigens to induce stable neutralizing antibodies and accompanying/concomitant T cell responses, thereby achieving protective immunization. In some aspects, a minimum vaccine dose is administered to induce stable neutralizing antibodies and accompanying/concomitant T cell responses, thereby achieving protective immunization. In one aspect, the RNA administered is in vitro transcribed RNA. For example, such RNA can be used to encode at least one antigen intended to produce an immune response in the mammal. Pathogenic antigens are peptides or protein antigens derived from pathogens associated with infectious diseases. In a particular aspect, pathogenic substances are peptides or protein antigens derived from VZV. Conditions and/or diseases that can be treated with RNA disclosed herein include, but are not limited to, those conditions and/or diseases caused and/or affected by viral infection. Such viruses include, but are not limited to, VZV.

As used herein, "prevent" or "prevention" when used in conjunction with the occurrence of a disease, disorder, and/or condition refers to reducing the risk of developing the disease, disorder, and/or condition and/or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered accomplished when the onset of a disease, disorder, or condition has been delayed for a predetermined period of time.

As will be understood from the context, the "risk" of a disease, illness and/or condition refers to the possibility that a specific subject will suffer from the disease, illness and/or condition. In some aspects, risk is expressed as a percentage. In some aspects, risk is, at least or at most 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% until 100%. In some aspects, risk is expressed as the risk relative to the risk associated with a reference sample or a reference sample group. In some aspects, a reference sample or a reference sample group has a known risk of a disease, illness, condition and/or event. In some aspects, a reference sample or a reference sample group is from a subject similar to a specific subject. In some aspects, risk can reflect one or more genetic attributes, such as it can make a subject tend to suffer from (or not suffer from) a specific disease, illness and/or condition. In some aspects, the risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. Susceptible to: A subject who is "susceptible to" a disease, disorder, and/or condition is a subject whose risk of suffering from the disease, disorder, and/or condition is higher than that of members of the general public. In some aspects, a subject who is susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some aspects, a subject who is susceptible to a disease, disorder, and/or condition may present symptoms of the disease, disorder, and/or condition. In some aspects, a subject who is susceptible to a disease, disorder, and/or condition may not present symptoms of the disease, disorder, and/or condition. In some aspects, a subject who is susceptible to a disease, disorder, and/or condition will suffer from the disease, disorder, and/or condition. In some aspects, a subject who is susceptible to a disease, disorder, and/or condition will not suffer from the disease, disorder, and/or condition.

The terms "protein", "polypeptide" or "peptide" are used as synonyms herein and refer to a polymer of amino acid monomers, such as a molecule comprising at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, heterologs, homologues, fragments and other equivalents, variants and analogs of the foregoing. Polypeptides may be unimolecular or may be multimolecular complexes, such as dimers, trimers or tetramers. Proteins contain one or more peptides or polypeptides and may be folded into a 3-dimensional form, which the protein may require to exert its biological function.

As used herein, the term "wild type" or "WT" or "native" refers to the endogenous form of a molecule that occurs naturally in an organism. In some aspects, a wild type form of a protein or polypeptide is employed, whereas in other aspects of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above are used interchangeably.

A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, in particular its amino acid sequence, is altered relative to a wild-type protein or polypeptide. In some aspects, a modified protein or polypeptide/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 particularly contemplated that a modified protein or polypeptide/variant protein or polypeptide may be altered relative to one activity or function, but retain wild-type activity or function in other aspects, such as immunogenicity. When a protein is specifically mentioned herein, it generally refers to a natural (wild-type) or recombinant (modified) protein. Proteins may be isolated directly from natural organisms, produced by recombinant DNA/exogenous expression methods, produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In particular aspects, there are isolated nucleic acid segments and recombinant vectors that incorporate a nucleic acid sequence encoding a polypeptide (e.g., an antigen or fragment thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, and this generally refers to a polypeptide produced by a nucleic acid molecule that has been manipulated in vitro or a polypeptide that is a replication product of such a molecule.

The term "fragment" with reference to an amino acid sequence (peptide or protein) refers to a portion of an amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and/or the C-terminus. A shortened fragment at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame at the 3' end without an open reading frame. A shortened fragment at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame at the 5' end without an open reading frame, as long as the truncated open reading frame comprises a start codon for initiating 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 an amino acid sequence. In the present disclosure, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least, at most, exactly, or any two of the following sequence identities to the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

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

As used herein in the context of molecules (e.g., nucleic acids, proteins, or small molecules), the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but is structurally different from the reference molecule, such as being different in the presence or absence of one or more chemical moieties or in the content of one or more chemical moieties compared to a reference entity. In some aspects, a variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity between it and the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural components. By definition, a variant is a different molecule that shares one or more such characteristic structural components with a reference molecule but is different from a reference molecule in at least one aspect. In some aspects, a variant polypeptide or nucleic acid may be different from a reference polypeptide or nucleic acid due to one or more differences in amino acid or nucleotide sequence and/or due to one or more differences in a chemical moiety (e.g., carbohydrate, lipid, phosphate group) as a covalent component of a polypeptide or nucleic acid (e.g., connected to a polypeptide or nucleic acid backbone). In some aspects, variant polypeptides or nucleic acids exhibit at least, at most, exactly the following overall sequence identity or overall sequence identity between any two of the following with reference polypeptides or nucleic acids: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%. In some aspects, variant polypeptides or nucleic acids do not share at least one characteristic sequence component with reference polypeptides or nucleic acids. In some aspects, reference polypeptides or nucleic acids have one or more biological activities. In some aspects, variant polypeptides or nucleic acids share one or more of the biological activities of reference polypeptides or nucleic acids. In some aspects, variant polypeptides or nucleic acids do not have one or more of the biological activities of reference polypeptides or nucleic acids. In some aspects, variant polypeptides or nucleic acids exhibit a reduction in one or more biological activity levels compared to reference polypeptides or nucleic acids. In some aspects, if the amino acid or nucleotide sequence of the polypeptide or nucleic acid of interest is consistent with the amino acid or nucleotide sequence of the reference but has a few sequence changes at a specific position, it is considered a "variant" of the reference polypeptide or nucleic acid. Preferably, the variant polypeptide or nucleic acid sequence has at least one modification compared to the reference polypeptide or nucleic acid sequence, for example, 1 to about 20 modifications. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to the reference polypeptide or nucleic acid sequence. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3% or about 2% of the residues in the variant are substituted, inserted or deleted compared to the reference. Typically, the variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2 or about 1) of substituted, inserted or deleted functional residues (e.g., residues involved in a specific biological activity) relative to the reference. In some aspects, the variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. In some aspects, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and typically less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some aspects, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion compared to the reference, and in some aspects comprises no additions or deletions.

In some aspects, 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 the purposes of the present disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) comprises an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant, and/or an amino acid substitution variant. A "variant" of a nucleotide sequence comprises 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, configurations, isoforms, allelic variants, species variants, and species homologs, especially those that occur in nature. The term "variant" particularly includes fragments of an amino acid or nucleic acid sequence.

Changes can be introduced into nucleic acid by mutation, thereby causing changes in the amino acid sequence of the polypeptide (e.g., antigen or antibody or antibody derivative) encoded by the nucleic acid. Mutation can be introduced using any technology known in the art. In one aspect, one or more specific amino acid residues are changed using, for example, a site-directed mutagenesis scheme. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis scheme. In some aspects, no matter how it is performed, mutant polypeptides can be expressed and screened for desired properties.

Mutations can be introduced into nucleic acids without significantly changing the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions that cause amino acid substitutions at non-essential amino acid residues can be performed. Alternatively, one or more mutations that selectively change the biological activity of the polypeptide encoded by the nucleic acid can be introduced into the nucleic acid. For example, mutations can quantitatively or qualitatively change biological activity. Examples of quantitative changes include increasing, decreasing or eliminating activity. Examples of qualitative changes include changing the antigenic specificity of the antibody.

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

The terms "% identical", "% identity" or similar terms are particularly intended to refer to the percentage of identical nucleotides or amino acids in the optimal alignment between the compared sequences. This percentage is purely statistical, and the differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the compared sequences. The comparison of two sequences is usually performed by comparing the sequences with respect to segments or "comparison windows" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group). In some aspects, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, as available on the National Center for Biotechnology Information (NCBI) website.

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

In some aspects, the degree of similarity or identity is given for at least, at most, just below, or any region between the following of the entire length of the reference sequence: about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given for at least, at most, just below, or any region between the following of the following number of nucleotides: about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides, in some aspects, continuous nucleotides. In some aspects, the degree of similarity or identity is given for the entire length of the reference sequence.

Homologous amino acid sequences can exhibit at least, at most, exactly, or any two of the following identities of amino acid residues: 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity. In one aspect, homologous amino acid sequences exhibit at least 95% identity of amino acid residues. In one aspect, homologous amino acid sequences exhibit at least 98% identity of amino acid residues. In one aspect, 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 refers to any fragment or variant that exhibits one or more functional properties that are identical or similar to the amino acid sequence from which it is derived, for example, it is functionally equivalent. With respect to antigens or antigenic sequences, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers in particular to a variant molecule or sequence comprising an amino acid sequence that has one or more amino acid changes compared to the parent molecule or sequence and is still able to achieve one or more functions of the parent molecule or sequence, such as inducing an immune response. In one aspect, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change 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 source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, it will be understood by those of ordinary skill in the art that an antigen suitable for use herein may be altered so that it differs in sequence from the naturally occurring sequence or native sequence from which it is derived while retaining the desired activity of the native sequence.

In the present disclosure, vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector can be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence comprising an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, transfer vectors. A vector can be an RNA vector or a DNA vector. In some aspects, the vector is a DNA molecule. In some aspects, the vector is a plasmid vector. In some aspects, the vector is a viral vector. Typically, an expression vector will contain the desired coding sequence and appropriate other sequences for expressing an operably connected coding sequence in a specific host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in an in vitro expression system. Cloning vectors are typically used for engineering and amplification of a desired fragment (typically a DNA fragment), and may lack the functional sequences required for expressing the desired fragment.

As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be an immunogenic composition. In some aspects, the active agent is present in a treatment regimen in an amount suitable for administration as a unit dose, which exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some aspects, the pharmaceutical composition may be specially formulated for parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection in the form of, for example, 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 produce, for example, an immune response to a disease-related (e.g., pathogenic) agent (e.g., a virus). In some aspects, vaccination may be administered before, during, and/or after exposure to a disease-related agent and in some aspects shortly before, during, and/or after exposure to the disease-related agent. In some aspects, vaccination includes multiple administrations of a vaccine composition appropriately spaced apart in time. In some aspects, vaccination produces an immune response to an infectious agent. In some aspects, vaccination produces an immune response to a tumor; in some such aspects, vaccination is "personalized" because it is partially or completely directed to an epitope (e.g., which may be or include one or more new epitopes) determined to be present in a tumor of a particular individual.

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

As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some aspects, two or more regimens may be administered simultaneously; in some aspects, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any dose of the second regimen); in some aspects, such agents are administered with overlapping dosing regimens. In some aspects, the "administration" of a combination therapy may involve administering one or more agents or modalities to a subject receiving other agents or modalities in combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or not even necessarily administered simultaneously), but in some aspects, two or more agents or their active portions may be administered together in a combined composition or even in a combined compound form (e.g., as part of a single chemical complex or covalent entity).

Those skilled in the art will appreciate that the term "dosage regimen" can be used to refer to a group of unit administrations (usually more than once) administered to a subject individually, which are usually separated by time periods. In some aspects, a given therapeutic agent has a recommended dosing regimen, which may involve one or more administrations. In some aspects, a dosing regimen includes multiple administrations, each of which is separated from other administrations in time. In some aspects, individual administrations are separated from each other by time periods of the same length; in some aspects, a dosing regimen includes multiple administrations and at least two different time periods separating individual administrations. In some aspects, all administrations within a dosing regimen have the same unit dosage. In some aspects, different administrations within a dosing regimen have different amounts. In some aspects, a dosing regimen includes the first administration of a first dosage, followed by one or more additional administrations of a second dosage that is different from the first dosage. In some aspects, a dosing regimen includes the first administration of a first dosage, followed by one or more additional administrations of a second dosage that is the same as the first dosage. In some aspects, a dosing regimen is associated with a desired or beneficial result (e.g., a therapeutic dosing regimen) when administered across a related population.

II. Varicella-zoster virus (VZV)

The present disclosure provides an RNA molecule (e.g., an RNA polynucleotide) comprising at least one open reading frame encoding a varicella zoster virus (VZV) polypeptide. The present disclosure further provides an immunogenic composition comprising at least one RNA molecule encoding a VZV polypeptide, the at least one RNA molecule being complexed with one or more lipids, encapsulated in one or more lipids, or formulated with one or more lipids and forming a lipid nanoparticle (LNP).

Varicella zoster virus (VZV), also known as human herpes virus 3 (HHV-3), is a human pathogen that causes chickenpox or chickenpox in children and later relapses into herpes zoster or shingles. VZV has an inner shell that surrounds a linear double-stranded DNA genome. The shell is surrounded by an outer coat with glycoproteins, and the outermost layer is a lipid-rich envelope with glycoproteins. Glycoproteins have a variety of functions, from DNA replication or shell assembly to interaction with cell surface molecules and auxiliary fusion into the plasma membrane. For example, glycoprotein E is an integral membrane protein that is considered important for T cell infection and cell-to-cell spread of the virus. VZV shows tropism to neurons and T cells.

After primary infection with VZV (e.g., varicella or "fowl 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. Deficiency of cell-mediated immunity (e.g., advanced age, immunocompromised conditions) is a risk factor for reactivation. Reactivation causes VZV replication and translocation to the skin, which may be expressed as herpes zoster (HZ).

Shingles most often presents as a painful, unilateral vesicular rash that is usually confined to one dermatome or to several connected dermatomes. Within days of the onset of the rash, groups of blisters, bullae, or pustules may appear; these lesions contain VZV and are considered contagious. The characteristic pain of shingles includes burning or numbness, itching, or tenderness to touch. Many people experience a prodromal pain 2 to 3 days before the rash appears. In immunocompetent individuals, the lesions crust over within 7 to 10 days and are no longer contagious after the crust has formed.

The most common complication of herpes zoster is postherpetic neuralgia (PHN), and up to 15% of individuals with herpes zoster develop PHN. PHN is a significant pain in the area infected by herpes zoster after the rash has crusted. Older age and prodromal symptoms are considered as risk factors for PHN. Other complications of herpes zoster include ocular complications (herpes zoster oculata or herpetic keratitis, acute retinal necrosis), neurological complications (herpes zoster oticus, meningitis, encephalitis, myelitis, peripheral motor neuropathy, Guillain-Barré syndrome (Guillan-Barré syndrome) and stroke) and secondary bacterial skin and soft tissue infections.

The VZV genome encodes at least 71 unique proteins (ORF0 to ORF68) and three additional open reading frames (ORF69 to ORF71) that replicate earlier (ORF64 to 62, respectively). The encoded proteins form the structure of the virus particles, 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 play a role in different stages of the viral replication cycle. The most abundant glycoprotein found in infected cells and mature virions is glycoprotein E (gE, ORF 68), which is the main 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 contributes to the endocytosis of the two glycoproteins and guides them to the trans-Golgi network (TGN), where the final viral envelope is obtained. VZV gE is a type I membrane protein of 623 amino acids encoded by open reading frame 68 (ORF68), and is the most abundant viral glycoprotein expressed on the surface of cells infected with VZV. Glycoprotein I (gI) is required in TGN for effective membrane fusion during VZV encapsulation and VZV replication. It was found that VZV gE and gI are compounded together on the surface of infected host cells. Glycoprotein B (ORF 31) is bound to neutralizing antibodies, which is the second most common glycoprotein and is considered to play a role in virus entry. Glycoprotein H is considered to have a fusion function that promotes the intercellular spread of the virus. Antibodies to gE, gB and gH are ubiquitous after natural infection and after vaccination, and have shown in vitro neutralization virus activity. As used herein, the term "varicella zoster virus" or "VZV" is not limited to any particular viral strain or variant.

In some aspects, the RNA molecule comprises an open reading frame encoding a VZV antigen. In some aspects, the VZV antigen is a VZV polypeptide. In some aspects, 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 aspects, 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 aspect, the RNA molecule encodes a VZV gE polypeptide. In some aspects, the VZV polypeptide comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more) VZV polypeptides.

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

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

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

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

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

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

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

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

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

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

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

In some aspects, the RNA molecule encodes a A VZV gE polypeptide having an amino acid sequence of any one of 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, the individual sequences of which are incorporated herein by reference. In some aspects, the RNA molecule encodes a polypeptide comprising a polypeptide according to The VZV gE polypeptide (ORF68) having the amino acid sequence of Accession No. AH009994.2 or a fragment or variant thereof, the sequence of which is incorporated herein by reference.

In some aspects, the RNA molecule encodes a VZV polypeptide of Table 1 (see Example 7). In some aspects, the RNA molecule encodes a VZV gE polypeptide comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 11, or a fragment or variant thereof. In some aspects, the VZV gE polypeptide may have at least, at most, exactly, or any two of the following identities to any of the amino acid sequences of Table 1, such as to any one of SEQ ID NOs: 1 to 11: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the VZV gE polypeptide consists of any one of the amino acid sequences of Table 1, such as any one of SEQ ID NOs: 1-11.

In some aspects, the RNA molecule sequence is transcribed from a DNA nucleic acid sequence (DNA polynucleotide) of Table 2 (see Example 7). In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence of any one of SEQ ID NOs: 12 to 145, or a fragment or variant thereof. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence that may have at least, at most, exactly, or any two of the following identities with any one of the nucleic acid sequences of Table 2, such as any one of SEQ ID NOs: 12 to 145: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any one of the nucleic acid sequences of Table 2, eg, consisting of any one of SEQ ID NOs: 12 to 145.

In some aspects, the RNA molecule comprises an ORF comprising an RNA nucleic acid sequence (RNA polynucleotide) of Table 3 (see Example 7). In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any one of SEQ ID NOs: 146 to 279, or a fragment or variant thereof. In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence that may have at least, at most, exactly, or any two of the following identities with any one of the RNA nucleic acid sequences of Table 3, such as any one of SEQ ID NOs: 146 to 279: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence consisting of any one of the RNA nucleic acid sequences of Table 3, such as any one of SEQ ID NOs: 146 to 279.

In some aspects, the RNA molecule comprises a stabilized RNA. In some aspects, the RNA molecule comprises a nucleic acid sequence that at least one uridine is replaced by N1-methyl pseudouridine. In some aspects, the RNA molecule comprises a sequence that all uridines are replaced by N1-methyl pseudouridine (expressed as "Ψ"). In some aspects, the RNA molecule comprises an ORF containing a nucleic acid sequence of any one of SEQ ID NO:146 to 279, wherein all uridines are replaced by N1-methyl pseudouridine (expressed as "Ψ").

In some aspects, the RNA molecule comprises an open reading frame encoding a VZV polypeptide amino acid sequence that can have at least, at most, exactly, or any between 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of the VZV polypeptide sequences of SEQ ID NOs: 1 to 11 (Table 1), or other VZV polypeptides described herein. In some aspects, the RNA molecule comprises an open reading frame encoding a VZV polypeptide amino acid sequence that consists of any one of the VZV polypeptide sequences of SEQ ID NOs: 1 to 11 (Table 1), or other VZV polypeptides described herein.

In some aspects, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence that can have at least, at most, exactly, or any two of the following identities to any one of the nucleic acid sequences of SEQ ID NOs: 12 to 145 (Table 2), or other nucleic acids described herein: 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence consisting of any one of the nucleic acid sequences of SEQ ID NOs: 12 to 145 (Table 2), or other nucleic acids described herein.

In some aspects, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence that can have at least, at most, exactly, or any of the following identities with any of the nucleic acid sequences of SEQ ID NOs: 146 to 279 (Table 3) or other nucleic acids described herein: 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence that consists of any of the nucleic acid sequences of SEQ ID NOs: 146 to 279 (Table 3) or other nucleic acids described herein. In some aspects, the RNA molecule comprises an ORF comprising the nucleic acid sequence of any of SEQ ID NOs: 146 to 279 (Table 3), wherein all uridines have been replaced by N1-methyl pseudouridine (denoted as "Ψ").

III. RNA molecules

In some aspects, RNA molecules described herein are coding RNA molecules. Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some aspects, coding RNA molecules include at least one open reading frame (ORF) encoding at least one peptide or polypeptide. The open reading frame includes a codon sequence that can be translated into a peptide or protein. Coding RNA molecules can include one (monocistron), two (bicistrons) or more (polycistronic) OFRs, which can be codon sequences that can be translated into polypeptides or proteins 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 aspects, the RNA molecule is mRNA. Preferably, the RNA molecule of the present disclosure is mRNA. In some aspects, the RNA molecule is saRNA. In some aspects, the saRNA molecule may be a coding RNA molecule.

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

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

It will be appreciated by those skilled in the art that the term "codon optimization" refers to codon changes in the coding region of a nucleic acid molecule that reflect the typical codon usage of the host organism without changing the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some aspects, the coding region is codon optimized for optimal expression in a subject to be treated with the RNA polynucleotides 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 the cell. Therefore, the sequence of the RNA can be modified so that codons that can obtain frequently occurring tRNA molecules are inserted at the position of a "rare codon".

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

In some aspects, the RNA molecule includes from about 20 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500). 00, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000 0, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

In some aspects, the RNA molecule has at least, at most, exactly, or any number of nucleotides between about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 860, 880, 800, 810, 820, 830, 840, 860, 880, 800, 810 ,740,760,780,800,820,840,860,880,900,920,940,960,980,1000,1000,1200,1400,1600,1800,2000,2200,2400,2600,2800,3000,3200,340 0, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 62 00, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 10000, 12000, 14000, 1600 0. 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 4 0000, 42000, 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000 or 100000 nucleotides.

In some aspects, the RNA molecule comprises at least 100 nucleotides. For example, in some aspects, the length of the RNA is between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; between 7,000 and 25,000 nucleotides. In some aspects, the RNA molecule has at least, at most, exactly, or any number of nucleotides between about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1910, 1920, 2030, 2040, 2050, 2150, 2160, 2170, 2180, 2200, 2250, 2300, 2350, 2400, 25 00, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 300 0, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3 50 50, 5100, 5150, 5200, 5250, 5300, 5350, 5400, 5450, 5500, 5550, 5600, 5650, 5700, 5750, 5800, 5850, 7 100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900 , 7950, 8000, 8050, 8100, 8150, 8200, 8250, 8300, 8350, 8400, 8450, 8500, 8550, 8600, 8650, 8700, 8750, 8800, 8850, 8900, 8950, 9000, 9050, 9100, 9150, 9200, 9250, 9300, 9350, 9400, 9450, 9500, 9550, 9600, 9650, 9700, 9750, 9800, 9850, 9900, 995 0, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950 ,11000,11050,11100,11150,11200,11250,11300,11350,11400,11450,11500,11550,11600,1165 0, 11700, 11750, 11800, 11850, 11900, 11950, 12000, 12050, 12100, 12150, 12200, 12250, 12300, 12350, 12400, 12450, 12500, 12550, 12600, 12650 ,12700,12750,12800,12850,12900,12950,13000,13050,13100,13150,13200,13250,13300,1335 1400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, 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, RNA is or comprises messenger RNA (mRNA), which is associated with an RNA transcript encoding a polypeptide. In some aspects, the RNA disclosed herein comprises: a 5' end cap, which comprises a 5' end cap disclosed herein; a 5' untranslated region, which comprises a proximal end cap sequence (5'UTR), i.e., a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3'UTR); and/or a polyadenylic acid (poly A) sequence.

In some aspects, the RNA disclosed herein comprises the following components in the 5' to 3' direction: a 5' end cap comprising the 5' end cap disclosed herein; a 5' untranslated region comprising a end cap proximal sequence (5'UTR), i.e., a sequence encoding a protein (e.g., a polypeptide); a 3' untranslated region (3'UTR); and a poly A sequence.

A. Modified Nucleobases

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

In some aspects, the RNA molecule may include modified nucleotides. Non-limiting examples of modified nucleotides that may be included in the RNA molecule include pseudouridine, N1-methyl pseudouridine, 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-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-taurinemethyl-uridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-1 -pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, 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-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, any other modified uridine known in the art, or a combination thereof.

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

In some aspects, the RNA molecule comprises nucleotides modified with N1-methyl pseudouridine. In some aspects, the RNA molecule comprises nucleotides modified with pseudouridine.

In some aspects, at least one uridine of RNA is replaced by a modified nucleoside. In some aspects, each uridine of RNA is replaced by a modified nucleoside. In some aspects, RNA molecules include a sequence in which at least one uridine is replaced by N1-methyl pseudouridine. In some aspects, RNA molecules include a sequence in which all uridines are replaced by N1-methyl pseudouridine. N1-methyl pseudouridine is represented as "Ψ" in the sequence. As used herein, the term "uracil" describes a kind of core base that may appear in the nucleic acid of RNA. As used herein, the term "uridine" describes a kind of nucleoside that may appear in RNA. "Pseudouridine" is an example of a modified nucleoside, which is an isomer of uridine, in which uracil is connected to a pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.

In some aspects, the RNA molecule comprises at least one uridine replaced by N1-methylpseudouridine or pseudouridine. In some aspects, the RNA molecule comprises at least, at most, exactly, or any two of the following percentages of uridine replaced by N1-methylpseudouridine or pseudouridine: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 10 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises a nucleic acid sequence in which all uridines are replaced with N1-methylpseudouridine or pseudouridine.

Modifications that may be present in RNA molecules further 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-methyladenosine); i6A (N6-isopentenyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); io6A (N6-(cis-hydroxyisopentenyl)adenosine); ms2io6A (2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine); g6A (N6-glycylcarbamoyladenosine); t6A (N6-threonylcarbamoyladenosine); acyl adenosine); 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-ribosyl adenosine (phosphorus) acid); 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-acetylcytidine); Acyl 2TO methylcytidine); k2C (lysidine); 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* (undermodified hydroxywybutosine); imG (wybutosine); mimG (methylguanosine); Q (Q nucleoside); oQ (epoxy Q nucleoside); galQ (galactosyl-Q nucleoside); manQ (mannosyl-Q nucleoside); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G* (archauridine); 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-(carboxy hydroxymethyl) uridine methyl ester); mcm5U (5-methoxycarbonylmethyl uridine); mcm5Um (S-methoxycarbonylmethyl-2-O-methyl uridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); nm5s2U (5-aminomethyl-2-thiouridine); mnm5U (5-methylaminomethyl uridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5-carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-O-methyl uridine); cmnm5U (5-carboxymethylaminomethyl uridine); cnmm5Um (5-carboxymethylaminomethyl-2-L-O-methyl uridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); 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-dimethylcytidine); methyladenosine); 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); tm5s2U (S-taurine methyl-2-thiouridine); imG-14 (4-demethylguanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, its 7-substituted derivatives, dihydrouracil, pseudouracil, 2-thio 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-hydroxyguanine, 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 aspects, the RNA molecules can include phosphoramidate, phosphorothioate, and/or methylphosphonate bonds.

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

In some aspects, the RNA molecules of the present disclosure include an open reading frame having a sequence modified by at least one codon. A sequence modified by a codon is a coding sequence that is different from the corresponding wild-type coding sequence in at least one codon (nucleotide triplet encoding an amino acid). A sequence modified by a codon can 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, such as human cells.

In some aspects, RNA molecules may include one or more structures and/or chemical modifications or changes that confer applicable properties to polynucleotides, and in some aspects, such applicable properties include substantial induction of the innate immune response of cells lacking the introduction of polynucleotides. As used herein, "structure" features or modifications to two or more connected nucleotides are inserted, deleted, replicated, reversed or randomized in RNA molecules without significant chemical modification of the nucleotides themselves. Because chemical bonds will inevitably break and reform to achieve structural modification, structural modification has chemical properties and is therefore chemically modified. However, structural modification will produce different nucleotide sequences. For example, polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified to "ATCCCG" from "ATCG". Here, dinucleotide "CC" has been inserted, thereby causing structural modification of polynucleotides.

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

In some aspects, the RNA molecule includes no modified nucleotides, e.g., no modified nucleobases, except for a 5' end cap (described further below) which may optionally include, e.g., 7-methylguanosine, and all nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C. In some aspects, the RNA may include a 5' end cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.

In some aspects, 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 aspects, non-coding RNA is a functional mRNA molecule that is not translated into a peptide or polypeptide. Non-coding RNA may include modified nucleotides as described herein. Preferably, the RNA molecule is mRNA.

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

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

In some aspects, the RNA molecules of the disclosure are lyophilized to be temperature stable.

B.5' end cap

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

In some aspects, the 5' end cap portion is a natural 5' end cap. "Natural 5' end cap" is defined as an end cap including a 7-methylguanosine connected to the 5' end of the mRNA molecule via a 5' to 5' triphosphate bond. In some aspects, the guanosine nucleoside included in the 5' end cap can be modified, for example, by methylation at one or more positions (e.g., 7 positions) on the base (guanine) and/or by methylation at one or more positions of ribose. In some aspects, the guanosine nucleoside included in the 5' end cap includes 3'O methylation (3'OMeG) at the ribose. In some aspects, the guanosine nucleoside included in the 5' end cap includes methylation (m7G) at the 7 positions of guanine. In some aspects, the guanosine nucleoside included in the 5' end cap includes methylation at the 7 positions of guanine and 3'O methylation (m7(3'OMeG)) at the ribose. The 5' end cap can be incorporated during RNA synthesis (e.g., co-transcriptional capping), or can be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some aspects, co-transcriptional capping with the end caps disclosed herein is compared to co-transcriptional capping with an appropriate reference comparator, and the capping efficiency of the RNA is improved. In some aspects, improving the capping efficiency can increase the translation efficiency and/or translation rate of the RNA, and/or increase the expression of the encoded polypeptide. In some aspects, capping is performed after purification of the RNA molecule (e.g., tangential flow filtration).

In some aspects, the RNA described herein comprises a 5' end cap or a 5' end cap analog, such as end cap 0, end cap 1 or end cap 2. In some aspects, the RNA provided does not have an uncapped 5'-triphosphate. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide. In some aspects, the 5' end cap portion is a 5' end cap analog. In some aspects, RNA can be capped with a 5' end cap analog. The end cap structure includes but is not limited to 7mG (5') ppp (5') N, pN2p (end cap 0) and 7mG (5') ppp (5') N1mpNp (end cap 1). In some aspects, the RNA described herein comprises end cap 0. End cap 0 is an N7-methylguanosine connected to a 5' nucleotide via a 5' to 5' triphosphate bond, which is commonly referred to as an m7G end cap or m7Gppp. In cells, the end cap 0 structure is crucial for the efficient translation of the mRNA carrying the end cap. Additional methylation at the 2'0 position of the starting nucleotide creates end cap 1, or is referred to as m7GpppNm, where Nm represents any nucleotide with 2'O methylation. In some aspects, the RNA described herein comprises end cap 1, e.g., as described herein. In some aspects, the RNA described herein comprises end cap 2.

In some aspects, the end cap O structure includes a guanosine nucleoside (m7G) methylated at the 7 position of guanine. In some aspects, the end cap O structure is connected to RNA via a 5' to 5' triphosphate bond, and is also referred to herein as m7Gppp or m7G(5')ppp(5'). The 5' end cap can be methylated with the structure m7G(5')ppp(5')N (end cap O structure) or its derivatives, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' end cap, typically the 5' end of the mRNA. Exemplary enzymatic reactions for capping may include the use of a vaccinia virus capping enzyme (VCE) including mRNA triphosphatase, guanylyl transferase, and guanine-7-methyltransferase, which catalyzes the construction of the N7-monomethylated end cap O structure. The end cap O structure plays an important role in maintaining the stability and translation efficacy of RNA molecules.

The 5' end cap of the RNA molecule can be further modified by a 2'-O-methyltransferase, which causes the generation of an end cap 1 structure (m7Gppp[m2'-O]N), which can further increase translation efficiency. In some aspects, the end cap 1 structure includes a guanosine nucleoside (m7G) methylated at the 7 position of guanine and the first nucleotide (2'OmeN ₁ ) methylated by 2'O in the RNA. In some aspects, the end cap 1 structure is connected to the RNA via a 5' to 5' triphosphate bond, and is also referred to herein as m7Gppp(2'OMeN ₁ ) or m7G(5')ppp(5')(2'OMeN ₁ ). In some aspects, N ₁ is selected from A, C, G or U. In some aspects, N ₁ is A. In some aspects, N ₁ is C. In some aspects, N ₁ is G. In some aspects, N ₁ is U. In some aspects, the m7G(5')ppp(5')(2'OmeN ₁ ) endcap 1 structure comprises a second nucleotide N ₂ which is the endcap 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 aspects, N ₂ is A. In some aspects, N ₂ is C. In some aspects, N ₂ is G. In some aspects, N ₂ is U.

In some aspects, the end cap 1 structure comprises a guanosine nucleoside methylated at the 7 position of guanine (m7G) and one or more additional modifications (e.g., methylation on ribose) and the first nucleotide methylated by 2'O in the RNA. In some aspects, the end cap 1 structure comprises a guanosine nucleoside methylated at the 7 position of guanine, 3'O methylation at the ribose (m7(3'OMeG)) and the first nucleotide methylated by 2'O in the RNA (2'OMeN ₁ ). In some aspects, the end cap 1 structure is connected to the RNA via a 5' to 5' triphosphate bond and is also referred to herein as m7(3'OMeG)ppp(2'OMeN ₁ ) or m7(3'OMeG)(5')ppp(5')(2'OMeN ₁ ). In some aspects, N ₁ is selected from A, C, G or U. In some aspects, N ₁ is A. In some aspects, N ₁ is C. In some aspects, N ₁ is G. In some aspects, _N1 is U. In some aspects, the m7(3'OMeG)(5')ppp(5')( _2'OMeN1 ) endcap 1 structure comprises a second nucleotide _N2 which is the endcap proximal nucleotide at position 2 and is selected from A, G, C or U (m7(3'OMeG)(5')ppp(5')( _2'OmeN1 ) _N2 ). In some aspects, _N2 is A. In some aspects, _N2 is C. In some aspects, _N2 is G. In some aspects, _N2 is U.

In some aspects, the second nucleotide in the Cap 1 structure can comprise one or more modifications, such as methylation. In some aspects, the Cap 1 structure comprising the second nucleotide comprising a 2'O methylation is a Cap 2 structure.

In some aspects, RNA molecules can be enzymatically capped at the 5' end using vaccinia guanylyl transferase, guanosine triphosphate and S-adenosyl-L-methionine to obtain an end cap 0 structure. The reverse 7-methylguanosine end cap is added via a 5' to 5' triphosphate bridge. Alternatively, a 2'O-methyltransferase and vaccinia guanylyl transferase are used to obtain an end cap 1 structure, wherein, except for the end cap 0 structure, the 2'OH group on the penultimate nucleotide is methylated. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer agent. Non-limiting examples of 5' end cap structures are those structures that have enhanced binding, increased half-life, reduced sensitivity to 5' endonucleases, and/or reduced 5' decapping, particularly for end cap-bound polypeptides, compared to synthetic 5' end cap structures known in the art (or wild-type, natural or physiological 5' end cap structures).

For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a typical 5'-5' triphosphate bond between the 5' terminal nucleotide of the mRNA and the guanine end cap nucleotide, wherein the end cap guanine includes N7 methylation and the 5' terminal nucleotide of the mRNA includes a 2'-O-methyl group. Such a structure is referred to as an end cap 1 structure. Compared to other 5' end cap analog structures known in the art, for example, this end cap results in higher translational competence and cellular stability and reduced activation of cellular proinflammatory cytokines.

In some aspects, the 5' end cap comprises an end 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 aspects, the capping region may include a single end cap or a series of nucleotides that form an end cap. In this aspect, the length of the capping region may be 1 to 10, such as 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or 10 or less nucleotides. In this aspect, the length of the capping region is at least, at most, exactly the following number of nucleotides, or any number of nucleotides between the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, there are no end caps. In some aspects, the length of the first and second operational regions may be in the range of 3 to 40 nucleotides, such as 5 to 30, 10 to 20, 15, or at least 4, or 30 or less nucleotides; and in addition to the start and/or stop codons, it may also include one or more signal and/or restriction sequences. In some aspects, the length of the first and second operative regions is at least, at most, exactly, or any two of the following number of nucleotides: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides, and in addition to the start and/or stop codons, they may also include one or more signal and/or restriction sequences.

Other examples of 5'-end cap structures include, but are not limited to, a glyceryl group, an inverted deoxy abasic residue (moiety), a 4',5' methylene nucleotide, a 1-(β-D-erythrofuranosyl) nucleotide, a 4'-thionucleotide, a carbocyclic nucleotide, a 1,5-anhydrohexitol nucleotide, an L-nucleotide, an α-nucleotide, a modified base nucleotide, a threo-pentofuranosyl nucleotide, a non-cyclic 3',4'-sequenced nucleotide, a non-cyclic 3,4-dihydroxybutyl nucleotide, a non-cyclic 3,5 dihydroxypentyl nucleotide, a 3'-3' inverted nucleotide moiety, a 3'-3' inverted abasic moiety, a 3'-2' inverted nucleotide moiety, a 3'-2' inverted abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3' phosphorothioate, a phosphorodithioate, or a bridged or non-bridged methyl phosphate moiety.

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

In one aspect, the 5' capping structure comprises a modified 5' end cap 1 structure (m ⁷ G ⁺ m ^3' -5'-ppp-5'-Am). In one aspect, the 5' capping structure comprises (3'OMe)-m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG (TriLink BioTechnologies). This molecule is consistent with the natural RNA end cap structure in that it begins with a guanosine methylated at N7 and is linked to the first encoded nucleotide of the transcribed RNA (in this case, adenosine) via a 5' to 5' triphosphate bond. This guanosine is also methylated at the 3' hydroxyl of the ribose to mitigate possible reverse incorporation of the end cap molecule. The 2' hydroxyl of the ribose on the adenosine is methylated, thereby conferring the end cap 1 structure.

C. Untranslated region (UTR)

The 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 a corresponding region in an RNA polynucleotide (such as an mRNA molecule). The untranslated region (UTR) may be present at the 5' end (upstream) of the open reading frame (5'UTR) and/or at the 3' end (downstream) of the open reading frame (3'UTR).

In some aspects, UTR is derived from naturally abundant mRNA in specific tissues (e.g., lymphoid tissue) targeted by mRNA expression. In some aspects, UTR increases protein synthesis. Not bound by mechanism or theory, UTR can increase protein synthesis by increasing the retention time (information stability) of mRNA in translation polyribosomes and/or the rate (information translation efficiency) of ribosomes based on information initiation translation. Therefore, UTR sequences can prolong protein synthesis in a tissue-specific manner.

In some aspects, 5'UTR and 3'UTR sequences are derived computationally. In some aspects, 5'UTR and 3'UTR are derived from naturally abundant mRNA in tissues. The tissue may be, for example, liver, stem cells, or lymphoid tissue. Lymphoid tissue may include, for example, 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 any one of reticulocytes. In some aspects, 5'UTR and 3'UTR are derived from alphaviruses. In some aspects, 5'UTR and 3'UTR are from wild-type alphaviruses.

i.5′UTR

In some aspects, the RNA disclosed herein comprises a 5'UTR. The 5'UTR, if present, is located at the 5' end and starts with the transcription start site upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5' end cap, if present, for example, directly adjacent to the 5' end cap. The 5'UTR may contain various regulatory components, such as a 5' end cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation.

In some aspects, the 5'UTR disclosed herein comprises, for example, an end cap proximal sequence as disclosed herein. In some aspects, the end cap proximal sequence comprises a sequence adjacent to the 5' end cap. In some aspects, the end cap proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some aspects, the end cap structure comprises one or more polynucleotides of the end cap proximal sequence. In some aspects, the end cap structure comprises an m7 guanosine end cap and nucleotide +1 ( _N1 ) of the RNA polynucleotide. In some aspects, the end cap structure comprises an m7 guanosine end cap and nucleotide +2 ( _N2 ) of the RNA polynucleotide. In some aspects, the end cap structure comprises an m7 guanosine end cap and nucleotides +1 and +2 ( _N1 and _N2 ) of the RNA polynucleotide.

Those skilled in the art who read the present disclosure will appreciate that in some aspects, one or more residues of the end cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) can be included in the RNA by virtue of being included in an end cap entity (e.g., end cap 1 structure, etc.); or, in some aspects, at least some of the residues in the end cap proximal sequence can be added enzymatically (e.g., by a polymerase, such as T7 polymerase). For example, in certain exemplary aspects utilizing ( _m27,3' ^-O )Gppp( ^m2'-O )ApG end caps, the +1 and +2 residues are the ( _m27,3' ^-O )A and G residues of the end cap, and the +3, +4, and +5 residues are added by a polymerase (e.g., T7 polymerase).

In some aspects, the end cap proximal sequence comprises _N1 and/or _N2 of the end cap structure, wherein _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some aspects, _N1 is A. In some aspects, _N1 is C. In some aspects, _N1 is G. In some aspects, _N1 is U. In some aspects, _N2 is A. In some aspects, _N2 is C. In some aspects, _N2 is G. In some aspects, _N2 is U. In some aspects, the end cap proximal sequence comprises _N1 and _N2 and _N3 , _N4 , and _N5 of the end cap structure, wherein _N1 to _N5 correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some aspects, _N1 , _N2 , _N3 , _N4 , or _N5 are any nucleotides, such as A, C, G, or U. In some aspects _{, N1N2} includes any one of the following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, or UU. In some aspects, N ₁ N ₂ includes AG and N ₃ N ₄ N ₅ includes any of: AAA, ACA, AGA, AUA, AAG, AGG, ACG, AUG, AAC, ACC, AGC, AUC, AAU, ACU, AGU, AUU, CAA, CCA, CGA, CUA, CAG, CGG, CCG, CUG, CAC, CCC, CGC, CUC, CAU, CCU, CGU, CUU, GA A, 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 aspects, the end cap proximal sequence comprises _N1 and _N2 of the end cap structure and a sequence comprising: _A3A4X5 (SEQ ID NO: 307; wherein _X5 is A, G, C or U), wherein _N1 and _N2 are each independently selected from: A, C, G or _U. In some aspects, _N1 is _A and _N2 is G. In some aspects, _X5 is selected from A, C, G or U. In some aspects, _X5 is A. In some aspects, _X5 is C. In some aspects, _X5 is G. In some aspects, _X5 is U.

In some aspects, the end cap proximal sequence comprises _N1 and _N2 of the end cap structure and a sequence comprising: _C3A4X5 (SEQ ID NO: 308; wherein _X5 is A, G, C or U), wherein _N1 and _N2 are each independently selected from: A, C, G or _U. In some aspects, _N1 is _A and _N2 is G. In some aspects, _X5 is selected from A, C, G or U. In some aspects, _X5 is A. In some aspects, _X5 is C. In some aspects, _X5 is G. In some aspects, _X5 is U.

In some aspects, the end cap proximal sequence comprises _N1 and _N2 of the end cap structure and _{a sequence comprising X3Y4X5 ( SEQ} ID NO: 309; wherein _X3 or _X5 are each independently selected from A, G, C or U; and _Y4 is not C). In some aspects, _N1 and _N2 are each independently selected from: A, C, G or U. In some aspects, _N1 is A and _N2 is G. In some aspects, _X3 and _X5 are each independently selected from A, C, G or U. In some aspects, _X3 and/or _X5 are A. In some aspects, _X3 and/or _X5 are C. In some aspects, _X3 and/or _X5 are G. In some aspects, _X3 and/or _X5 are U. In some aspects, _Y4 is C. In other aspects, _Y4 is not C. In some aspects, _Y4 is A. In some aspects, _Y4 is G. In other aspects, _Y4 is not G. In some aspects, Y4 is U.

In some aspects, the end cap proximal sequence comprises _N1 and _N2 of the end cap structure and a sequence comprising _A3C4A5 (SEQ ID NO: 310). In some aspects, _{N1 and N2} are each independently selected from: A, C, _G or U. In some aspects, _N1 is A and _N2 is G.

In some aspects, the end cap proximal sequence comprises _N1 and _N2 of the end cap structure and a sequence comprising _A3U4G5 (SEQ ID NO: 311). In some aspects, _N1 and _N2 are each independently selected from: A, C _, G _or U. In some aspects, _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 aspects, the RNA disclosed herein comprises a hAg 5'UTR or a fragment thereof. 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: 312. In some aspects, the RNA disclosed herein comprises a hAg 5'UTR provided in SEQ ID NO: 312.

SEQ ID NO:312

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCC

In some aspects, the RNA disclosed herein comprises a hAg 5'UTR that is 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical 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

AAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCC

In one aspect, the DNA encoding the 5'UTR disclosed herein comprises a sequence having at least, at most, exactly, or any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 280. In one aspect, the DNA encoding the 5'UTR comprises the sequence of SEQ ID NO: 280. In one aspect, the RNA disclosed herein comprises a 5'UTR comprising a sequence having at least, at most, exactly, or any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the 5'UTR provided in any one of SEQ ID NOs: 281 to 282, wherein the transcribed 5' cap structure is underlined. In one aspect, the 5'UTR comprises the sequence of any one of SEQ ID NOs: 281 to 282, wherein the transcribed 5' cap structure is underlined.

SEQ ID NO:280 (DNA)

AG AATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC

SEQ ID NO:281 (RNA)

AG AAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC

SEQ ID NO:282 (RNA)

AG AAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCACAGACΨCAGAGAGAACCCGCCACC

ii.3'UTR

In some aspects, the RNA disclosed herein comprises a 3'UTR. The 3'UTR, if present, is located downstream of the open reading frame of the protein coding sequence, for example, downstream of the stop codon of the protein coding region. The 3'UTR is generally the portion of the mRNA between the protein coding sequence and the poly-A tail of the mRNA. Thus, in some aspects, the 3'UTR is upstream of the poly-A sequence, if present, for example, directly adjacent to the poly-A sequence. The 3'UTR may be involved in regulatory processes, including transcript cleavage, stability and poly-A, translation, and mRNA localization.

3'UTR can also include components that are not encoded in the template of the transcribed RNA but are added during maturation after transcription, such as a polyadenylic acid tail. The 3'UTR of mRNA is not translated into an amino acid sequence. In some aspects, the RNA disclosed herein includes a 3'UTR containing an F component and/or an I component. In some aspects, the 3'UTR or its proximal sequence includes a restriction site. In some aspects, the restriction site is a BamHI site. In some aspects, the restriction site is a Xhol site.

In some aspects, the RNA disclosed herein comprises a 3'UTR having at least, at most, exactly, or any between 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity 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

CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGG GUUGGUCAAUUUCGUGCCAGCCACACC

In one aspect, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least, at most, exactly, or any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to SEQ ID NO: 283. In one aspect, the DNA encoding the 5'UTR comprises the sequence of SEQ ID NO: 283. In one aspect, the RNA disclosed herein comprises a 3'UTR comprising a sequence having at least, at most, exactly, or any two of 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the 3'UTR provided in any one of SEQ ID NOs: 284 to 285 and 317 to 318. In one aspect, the 3'UTR comprises the sequence of any one of SEQ ID NOs: 284 to 285 and 317 to 318.

SEQ ID NO:283 (DNA)

CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTG GTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC

SEQ ID NO:284 (RNA)

CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCC AGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC

SEQ ID NO:285 (RNA)

CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCACΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGCACGCAGCAA ΨGCAGCΨCAAAACGCΨΨA

GCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨ

ΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACCCΨGGAGC

ΨAGC

SEQ ID NO:317 (RNA)

CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCG

AGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACC

UCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGC

CACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC

UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACC

SEQ ID NO:318 (RNA)

CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨACC

CCGAGΨCΨCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCACΨCA

CCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGCACGCAGCAAΨGCAGCΨCAAAACGCΨΨA

GCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨ

ΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨΨCGΨGCCAGCCACACC

D. Open reading frame (ORF)

The 5' and 3' UTRs are operably linked to an open reading frame (ORF), which may be a sequence of codons that can be 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 a start codon at its 5' end and the next region, such as a combination of three consecutive nucleotides (ATG or AUG) that typically encode the amino acid methionine, and its length is typically a multiple of 3 nucleotides. An open reading frame may terminate 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 can be terminated by 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), NO: 306), or any combination thereof. An open reading frame may be isolated, or it may be incorporated into a longer nucleic acid sequence, for example, into a vector or mRNA. An open reading frame may also be referred to as a "(protein) coding region" or "coding sequence".

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

In some aspects, the ORF encodes a nonstructural viral gene. In some aspects, the ORF further comprises one or more subgenomic promoters. In some aspects, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some aspects, the first RNA molecule does not include an ORF encoding any polypeptide of interest, and the second RNA molecule includes an ORF encoding a polypeptide of interest. In some aspects, the first RNA molecule does not include 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 aspects, the RNA molecule comprises at least one open reading frame encoding a VZV gE polypeptide.

E. Genes of interest

The RNA molecules described herein may include a gene of interest. The gene of interest encodes a polypeptide of interest. Non-limiting examples of polypeptides of interest include, for example, biologics, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, polypeptides encoded by the human genome that have not yet been identified for therapeutic indications but are useful in the field of research and exploration, or combinations thereof. The sequence of a specific gene of interest is readily available to those skilled in the art using public and private databases (e.g. ) identification.

In some aspects, the RNA molecule includes the coding region of the gene of interest. In some aspects, the gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or immunogenic fragment thereof. In some aspects, the antigenic polypeptide comprises an epitope from an antigen. In some aspects, the antigenic polypeptide comprises a plurality of different epitopes from an antigen. In some aspects, the antigenic polypeptide comprising a plurality of different epitopes from an antigen is polyepitopic. In some aspects, the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasite antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide.

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

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

In some aspects, the RNA molecule includes a coding region of a gene of interest (e.g., an antigen). In some aspects, the RNA molecule includes a coding region of a gene of interest (e.g., an antigen) derived from a pathogen associated with an infectious disease. In some aspects, the RNA molecule includes a coding region of a gene of interest (e.g., an antigen) derived from a varicella zoster virus (VZV).

In some aspects, the RNA molecule encodes a VZV gE protein or a fragment or variant thereof. In some aspects, the RNA molecule encodes a protein comprising VZV gE protein having an amino acid sequence of any of the following: 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, the individual sequences of which are incorporated herein by reference. In some aspects, the RNA molecule encodes a VZV gE protein having an amino acid sequence of any of the following: 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, the individual sequences of which are incorporated herein by reference. The VZV gE protein having the amino acid sequence of Accession No. AH009994.2, the sequence of which is incorporated herein by reference.

In some aspects, the RNA polynucleotides described herein or the compositions or medical preparations comprising them comprise the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides comprise sequences having at least 80% identity with the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides comprise sequences encoding polypeptides, which have at least 80% identity with the polypeptide sequences disclosed herein. In some aspects, the RNA polynucleotides described herein or the compositions or medical preparations comprising them are transcribed by DNA templates. In some aspects, the DNA templates for transcribing the RNA polynucleotides described herein comprise sequences complementary to the RNA polynucleotides. In some aspects, the genes of interest described herein are encoded by the RNA polynucleotides described herein comprising the nucleotide sequences disclosed herein. In some aspects, the RNA polynucleotides encode polypeptides having at least 80% identity with the polypeptide sequences disclosed herein. In some aspects, the polypeptides described herein are encoded by RNA polynucleotides, which are transcribed by DNA templates comprising sequences complementary to the RNA polynucleotides.

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

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

F. Polyadenylation tail

In some aspects, the RNA molecules disclosed herein include, for example, polyadenylic acid (poly A) sequences as described herein. In some aspects, the poly A sequence is located downstream of the 3'UTR, for example, adjacent to the 3'UTR. "Poly A tail" or "poly A sequence" refers to a sequence segment of continuous adenine residues that can be connected to the 3' end of the RNA molecule. The poly A sequence is known to those skilled in the art and can be attached to the 3'UTR in the RNA molecules described herein. The poly A tail can increase the half-life of the RNA molecule.

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

A DNA sequence encoding a poly A sequence (coding strand) is referred to as a poly A box. In some aspects, the poly A box present in the coding strand of the DNA consists essentially of dA nucleotides, but is interspersed with a random sequence of four nucleotides (dA, dC, dG, and dT). Such a random sequence may be at least, at most, exactly, or any number of nucleotides between the following: 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 nucleotides in length. Such boxes are disclosed in WO 2016/005324 A1, which is hereby incorporated by reference. Any poly A box disclosed in WO 2016/005324 A1 can be used in the present disclosure. The present disclosure encompasses a poly A box consisting essentially of dA nucleotides, but interspersed with a random sequence having an equal distribution of four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, which exhibits constant propagation of plasmid DNA in Escherichia coli (E. coli) at the DNA level and is still associated with beneficial properties for supporting RNA stability and translation efficiency at the RNA level. In some aspects, the poly A sequence contained in the RNA polynucleotide described herein consists essentially of adenosine nucleotides, but interspersed with a random sequence of four nucleotides (A, C, G, U). The length of such random sequences may be at least, at most, exactly, or any number of nucleotides between: 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 nucleotides.

In some aspects, no nucleotides other than adenosine nucleotides flank the poly A sequence at its 3' end, e.g., the 3' end of the poly A sequence is not masked by or followed by nucleotides other than adenosine.

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

The poly A sequence can have any length. In some aspects, the poly A tail can include a length of 5 to 300 nucleotides. In some aspects, the RNA molecule includes a poly A tail, which includes, is essentially composed of, or is composed 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 aspects, the poly(A) tail comprises, consists essentially of, or consists of at least, at most, exactly, or between any two of the following number of adenosine nucleotides: 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, 211, 212, 213, 214, 215 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 550, 560, 570, 580, 590, 595, 600, 610, 615, 620, 625, 630, 640, 650, 670, 680, 690, 701, 711, 712, 713, 714, 715 In this context, "consisting essentially of" means that the majority of the nucleotides in the poly A sequence (typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly A sequence) are adenosine nucleotides, but the remaining nucleotides are permitted to be nucleotides other than adenosine nucleotides, such as uridine, guanosine, or cytosine. In this context, "consisting of" means that all nucleotides in the poly A sequence (e.g., 100% of the number of nucleotides in the poly A sequence) are adenosine nucleotides.

In some aspects, the RNA molecule includes a poly A tail comprising a sequence of more than 30 adenosine nucleotides. In some aspects, the RNA molecule includes a poly A tail comprising about 40 adenosine nucleotides. In some aspects, the RNA molecule includes a poly A tail comprising about 80 adenosine nucleotides. In some aspects, the 3' poly A tail has a sequence segment of at least 10 continuous adenosine residues and at most 300 continuous adenosine residues. In some specific aspects, the RNA molecule includes about 40 continuous adenosine residues. In some aspects, the RNA molecule includes about 80 continuous adenosine residues. The poly A tail can play a key regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or super polyadenylation can signal RNA degradation. Some designs include about 40 adenosine nucleotides.

In some aspects, the poly(A) tail may be located within an RNA molecule or other nucleic acid molecule, such as in a vector, for example, in a vector that serves as a template for producing RNA (e.g., mRNA), for example, by transcription of the vector. In some aspects, the RNA molecule may not include a poly(A) tail.

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

SEQ ID NO:286 (DNA)

AAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:287 (RNA)

AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:288(RNA)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:315 (RNA)

AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO:316 (RNA)

AAAAAAAAAAAAAAAAAAAAAAAAAAAGCAΨAΨGACΨAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

G. Self-amplifying RNA (SARNA)

In some aspects, the RNA molecule may be a saRNA. "Self-amplifying RNA", "self-amplifying RNA" and "replicon" refer to RNA that is capable of self-replication. Self-amplifying RNA molecules can be produced by using replication components derived from, for example, alpha viruses and replacing structural viral polypeptides with nucleotide sequences encoding polypeptides of interest. Self-amplifying RNA molecules are typically positive-strand molecules that can be translated directly after delivery to cells, and this translation provides RNA-dependent RNA polymerases, which then produce antisense and sense transcripts from the delivered RNA. The delivered RNA causes the generation of multiple daughter RNA molecules. These daughter RNA molecules, as well as colinear subgenomic transcripts, can themselves be translated to provide in situ expression of the encoded gene of interest (e.g., viral antigens), or can be transcribed to provide other transcripts synonymous with the delivered RNA that is translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is that the number of introduced saRNA molecules is amplified, and thus the encoded gene of interest (e.g., viral antigens) becomes the main polypeptide product of the cell.

In some aspects, the self-amplifying RNA includes at least one or more genes, including any one of a viral replicase, a viral protease, a viral helicase, and other non-structural viral proteins or a combination thereof. In some aspects, the self-amplifying RNA may also include 5' and 3' end traction replication sequences, and optionally a heterologous sequence encoding a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter directing the expression of a heterologous sequence may be included in the self-amplifying RNA. Optionally, a 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 aspects, the self-amplifying RNA molecules described herein encode: (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule; and (ii) a polypeptide of interest, such as a viral antigen. In some aspects, the polymerase can be an alphavirus replicase, for example, including any of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof.

In some aspects, 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 aspects, the RNA may have additional (e.g., downstream) open reading frames, for example to encode other antigens or to encode auxiliary polypeptides.

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

In some aspects, the self-amplifying RNA molecule can encode a single polypeptide antigen, or optionally encode two or more polypeptide antigens, which are linked together (e.g., linked in series) in a manner that each of the sequences retains its identity when expressed in the form of an amino acid sequence. The polypeptide produced by the self-amplifying RNA can then be produced in the form of a fusion polypeptide, or engineered in a manner that produces a separate polypeptide or peptide sequence.

In some aspects, the self-amplifying RNA described herein can encode one or more polypeptide antigens including a series of epitopes. In some aspects, the self-amplifying RNA described herein can encode epitopes capable of inducing a helper T cell response or a cytotoxic T cell response or both.

IV. RNA transcription

In some aspects, RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, RNA can 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 vitro in a non-cell system to produce a synthetic RNA product for various applications, including, for example, the production of proteins or polypeptides. Cloning vectors can be used to produce transcripts. These cloning vectors are generally referred to as transcription vectors and are covered by the term "vector" according to the present disclosure. According to a specific aspect, the RNA used is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, in vitro transcription according to the present disclosure is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid (especially cDNA) and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

The synthetic IVT RNA product can be translated in vitro or directly introduced into the cell, and the product can be translated in the cell. In terms of RNA, the term "expression" or "translation" refers to the process of guiding the assembly of an amino acid sequence in the ribosome of a cell by an mRNA chain to produce a peptide or protein. Such synthetic RNA products include, for example, but not limited to, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long non-coding RNA molecules, ribozymes, aptamers, guide RNA molecules (e.g., guide RNA molecules for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, etc. The IVT reaction typically utilizes a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates) and an appropriate RNA polymerase as described and/or utilized herein.

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

In some aspects, the starting materials for IVT can include linearized DNA template, nucleotides, RNase inhibitors, pyrophosphatase, and/or T7 RNA polymerase. In some aspects, the IVT process is performed in a bioreactor. The bioreactor can include a mixer. In some aspects, nucleotides can be added to the bioreactor during the entire IVT process.

In some aspects, after the IVT process, one or more post-IVT reagents are added to the IVT mixture containing RNA in the bioreactor. Exemplary post-IVT reagents 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 aspects, after IVT, the post-IVT reagents are incubated with the mixture in the bioreactor. In some aspects, the bioreactor may contain at least, at most, exactly, or any number of liters of the IVT mixture of 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 more liters. The IVT mixture can have an RNA concentration of at least, at most, exactly, or between any two of 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 more RNA.

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

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

In some aspects, "ultrafiltration" and "diafiltration" both refer to membrane filtration processes. Ultrafiltration typically uses membranes with pore sizes of at least, at most, just, or any size between the following: 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO can be at least, at most, just below, or between any two of the following: 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 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa and 10000 kDa. It will be appreciated by those skilled in the art that, depending on the application, the filtration membrane can have different suitable materials, including, for example, polymers, cellulose, ceramics, etc. In some aspects, membrane filtration is more ideal for large volume purification processes.

In some aspects, ultrafiltration and diafiltration of the IVT mixture used to purify RNA can include: (1) direct flow filtration (DFF), also known as "dead-end" filtration, which applies feed flow perpendicular to the membrane surface and attempts to pass 100% of the fluid through the membrane; and/or (2) tangential flow filtration (TFF), also known as sweep flow filtration, in which the feed flow is passed parallel to the membrane surface, with a portion passing through the membrane (permeate) and the remainder (retentate) retained and/or recycled back to the feed storage tank.

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

A filter membrane with an appropriate MWCO can be selected for ultrafiltration during TFF. The MWCO of a TFF membrane determines which solutes can enter the filtrate through the membrane and which solutes are retained in the retentate. The MWCO of a TFF membrane can be selected so that substantially all solutes of interest (e.g., desired synthetic RNA species) are retained in the retentate, while non-desired components (e.g., excess ribonucleotides, small nucleic acid fragments (such as digested or hydrolyzed DNA templates), peptide fragments (such as digested proteins) and/or other impurities) are delivered to the filtrate. In some aspects, the retentate comprising the desired synthetic RNA species can be recycled to the feed tank to be refiltered in an additional cycle. In some aspects, the MWCO of a TFF membrane can be at least, at most, just equal to the following or between any two of the following: 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa or greater. In some aspects, the MWCO of the TFF membrane may be at least, at most, just equal to, or between any two of the following: 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa or greater. In some aspects, the MWCO of the TFF membrane may be about 250-350 kDa. In some aspects, the MWCO of the TFF membrane (e.g., a cellulose-based membrane) may be about 30-300 kDa; in some aspects, about 50-300 kDa, about 100-300 kDa, or about 200-300 kDa.

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

In some aspects, for ultrafiltration and/or diafiltration, the IVT mixture is filtered at a rate of at least, at most, exactly equal to, or between any two of 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 filter area/hour or more. The concentrated RNA solution can contain single-stranded RNA at a concentration of at least, at most, just below, or any between: 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL.

In some aspects, the bioburden of the concentrated RNA solution obtained by filtration to obtain the RNA product solution can also be reduced. Filtration for reducing bioburden can be performed using one or more filters. The one or more filters can include a filter having a pore size of at least, at most, exactly, or between any two of the following: 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.

As an example, reducing the bioburden may include draining a retentate tank containing a retentate obtained from ultrafiltration and/or diafiltration to obtain a retentate. Reducing the bioburden may include flushing a filtration system for ultrafiltration and/or diafiltration with a wash buffer solution to obtain a wash pool solution containing 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 a first 0.2 μm filter or another 0.2 μm filter.

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

V. RNA Encapsulation

The RNA in the RNA product solution can be encapsulated, and the RNA solution can further include at least one encapsulating agent. In one aspect, the encapsulating agent includes lipids, lipid nanoparticles (LNPs), lipid complexes, polymer particles, polymer complexes, and integral delivery systems, and combinations thereof.

In one aspect, the encapsulating agent is a lipid, and produces the RNA of lipid nanoparticle (LNP) encapsulation. It is not intended to be bound by any theory, it is believed that cations or cation ionizable lipids or lipidoid substances and/or cationic polymers are combined with nucleic acids to form aggregates, and this aggregation produces colloidally stable particles. Lipids can be naturally occurring lipids or synthetic lipids. However, lipids are generally biological substances. Biological lipids are well known in the art, and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenoids, lyso-lipids, glycosphingolipids, glycolipids, sulfolipids, lipids and polymerizable lipids with ether and ester-connected fatty acids and combinations thereof. Lipids are water-insoluble and extractable organic solvent-extractable substances. Compounds other than these compounds specifically described herein are understood to be lipids by those skilled in the art, and are covered by the compositions and methods of the present disclosure. Lipid components and non-lipids can be covalently or non-covalently linked to each other.

In some aspects, LNPs can be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular RNases, and/or can be engineered to deliver RNA systemically to target cells. In some aspects, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when RNA molecules are administered intravenously to a subject in need. In some aspects, such LNPs may be particularly useful for delivering RNA molecules (e.g., saRNA, mRNA) when RNA molecules are administered intramuscularly to a subject in need.

In one aspect, the concentration of RNA in the RNA solution is <1 mg/mL. In another aspect, the concentration of RNA is at least about 0.05 mg/mL. In another aspect, the concentration of RNA is at least about 0.5 mg/mL. In another aspect, the concentration of RNA is at least about 1 mg/mL. In another aspect, the concentration of RNA is about 0.05 mg/mL to about 0.5 mg/mL. In another aspect, the concentration of RNA is at least 10 mg/mL. In another aspect, the concentration of RNA is at least 50 mg/mL. In some aspects, the concentration of RNA is at least, at most, just below, or between any two of the following: 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 higher.

The present disclosure provides RNA solutions and lipid formulation mixtures or compositions thereof, comprising at least one RNA encoding, for example, an antigen (e.g., a VZV polypeptide), the at least one RNA being complexed with, encapsulated in, and/or formulated with one or more lipids, and forming lipid nanoparticles (LNPs), liposomes, lipid complexes, and/or nanoliposomes. In some aspects, the compositions comprise lipid nanoparticles.

Lipid nanoparticle or LNP refers to any form of particle produced when cationic lipid and optional one or more other lipids are for example merged in an aqueous environment and/or in the presence of RNA. In some respects, lipid nanoparticle is included in a formulation, which can be used for delivering an active agent or therapeutic agent (such as nucleic acid (such as mRNA)) to a target site of interest (such as cell, tissue, organ, tumor, etc.). In some respects, the lipid nanoparticle of the present disclosure comprises nucleic acid. Such lipid nanoparticles generally comprise cationic lipid and one or more excipients, such as one or more neutral lipids, charged lipids, steroids, polymer conjugated lipids or their combination. In some respects, an active agent or therapeutic agent (such as nucleic acid (such as mRNA)) can be encapsulated in the lipid portion of lipid nanoparticle or in an aqueous space surrounded by some or all lipid portions of lipid nanoparticles, thereby protecting it from other undesirable effects caused by the mechanism of enzymatic degradation or host organism or cell, such as adverse immune response. Nucleic acid (such as mRNA) or its part can also be associated and compounded with lipid nanoparticle. The lipid nanoparticles may comprise any lipid capable of forming a particle that is attached to a nucleic acid or that encapsulates one or more nucleic acids.

In some aspects, the RNA molecules (e.g., saRNA, mRNA) provided can be formulated with LNP. In some aspects, the average diameter of the lipid nanoparticles can be about 1 to 500 nm. In some aspects, the average diameter of the lipid nanoparticles is about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or at least, at most , exactly or between any two of the following: 30nm, 35nm, 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm, and is substantially non-toxic. The term "average diameter" refers to the average hydrodynamic diameter of the particles measured by dynamic laser scattering (DLS) with data analysis using the so-called cumulant algorithm, the results of which provide the so-called Z-average of the length dimension and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Herein, the "average diameter," "diameter," or "size" of the particles is used synonymously with this Z-average value.

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 LNPs may exhibit a polydispersity index of at least, at most, just below, or between any two of 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. In some aspects, the polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulant analysis as mentioned in the definition of "mean diameter". Under certain prerequisites, it can be considered a measure of the size distribution of the nanoparticle population.

In some aspects, nucleic acid (e.g., RNA molecule) is resistant to nuclease degradation in aqueous solution when present in the LNP provided. In some aspects, LNP is a lipid nanoparticle targeting the liver. In some aspects, LNP is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., cationic lipids described herein). In some aspects, cationic LNP can include at least one cationic lipid, at least one polymer-conjugated lipid and at least one auxiliary lipid (e.g., at least one neutral lipid).

In certain aspects, RNA solutions and lipid formulation mixtures or compositions thereof may have the following amounts, at least the following amounts, or at least, at most, exactly the following amounts, or any amount between the following amounts of a particular lipid, lipid-type or non-lipid component, such as a lipid-like substance and/or a cationic polymer or adjuvant, antigen, peptide, polypeptide, sugar, nucleic acid, or other substance disclosed herein or as would be known to one skilled in the art: about 1%, about 2%, about 3%, about 4%, about 6%, 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 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 , 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%.

LNP described herein can use the method preparation of wide range, and described method can relate to obtain colloid and mix this colloid and nucleic acid to obtain nucleic acid particle by at least one cation or cation ionizable lipid or lipidoid material and/or at least one cationic polymer.Term " colloid " as used herein is about a kind of uniform mixture that dispersed particles can not precipitate out.Insoluble particles in mixture are tiny, and its particle diameter is between 1 and 1000 nanometers.Mixture can be referred to as colloid or colloidal suspension.Sometimes, term " colloid " only refers to the particle in mixture rather than whole suspension.

In order to prepare a colloid comprising at least one cationic or cationic ionizable lipid or lipidoid material and/or at least one cationic polymer, conventional methods for preparing liposome vesicles and appropriately adapted can be applied herein. The most commonly used methods for preparing liposome vesicles have the following basic stages: (i) lipids are dissolved in an organic solvent, (ii) the resulting solution is dried, and (iii) the dried lipids are hydrated (using various aqueous media). In the film hydration method, the lipids are first dissolved in a suitable organic solvent and dried to produce a film at the bottom of the flask. The resulting lipid film is hydrated using an appropriate aqueous medium to produce a liposome dispersion. In addition, an additional size reduction step may be included.

Reverse phase evaporation is an alternative method to membrane hydration for the preparation of liposome vesicles, which involves the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. Brief sonication of this mixture is required to homogenize the system. Removal of the organic phase under reduced pressure yields a milky white gel, which subsequently becomes a liposome suspension.

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

The term "extruding" or "extrusion" refers to the production of particles having a fixed cross-sectional profile. In particular, it refers to the reduction of the size of particles, wherein the particles are forced through a filter with defined pores.

Other methods having organic solvent-free properties may also be used to prepare colloids according to the present disclosure.

In some aspects, RNA encapsulated through LNP can be produced by rapidly mixing RNA solution (e.g., RNA product solution) described herein with lipid formulation (e.g., at least one cationic lipid and optionally one or more other lipid components contained in an organic solvent) described herein under conditions of triggering a solubility mutation of the lipid component, and the solubility mutation causes lipid to self-assemble in the form of LNP. In some aspects, suitable buffers 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 less 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 greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, the characteristic of the cationic lipid is selected so that the initial formation of the particle occurs by associating with the oppositely charged backbone of the nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which in some aspects can result in, for example, a 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 aspects, the nucleic acid, when present in lipid nanoparticles, is resistant to nuclease degradation in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods for preparing the same are disclosed in, for example, U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Publication Nos. WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.

Some aspects described herein are about compositions, methods and uses involving more than one, e.g., 2, 3, 4, 5, 6 or even more nucleic acid species (such as RNA species). In LNP formulations, it is possible to separately formulate each nucleic acid species into individual LNP formulations. In this case, each individual LNP formulation will contain a nucleic acid species. Individual LNP formulations can exist in the form of separate entities, such as in separate containers. Such formulations can be obtained by separately providing each nucleic acid species (usually each in the form of a solution containing nucleic acids) and suitable cations or cationically ionizable lipids or lipidoid materials and cationic polymers that allow the formation of LNPs. Individual particles will only contain the specific nucleic acid species (individual particle formulations) provided when forming particles.

In some aspects, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Individual pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the present disclosure can be obtained by separately forming individual LNP formulations as described above, followed by a step of mixing the individual LNP formulations. By the mixing step, a formulation comprising a mixed population of LNPs containing nucleic acids can be obtained. Individual LNP populations can be together in one container, which comprises a mixed population of individual LNP formulations.

Alternatively, different nucleic acid species may be formulated together as a combined LNP formulation. Such formulations can be obtained by providing a combined formulation of different RNA species (usually a combined solution) and suitable cationic or cationic ionizable lipids or lipidoid materials and cationic polymers that allow the formation of LNPs. In contrast to mixed LNP formulations, combined LNP formulations will typically contain LNPs comprising more than one RNA species. In combined LNP compositions, different RNA species are typically present together in a single particle.

A. Cationic polymers

In view of the high chemical flexibility of polymeric substances, it is usually used for delivery based on nanoparticles. Usually, cationic substances are used to make negatively charged nucleic acid electrostatically condense into nanoparticles. This positively charged group is usually composed of ammonia that changes in the pH range between 5.5 and 7.5 by protonation state, and it is believed that this change can cause the ion imbalance that causes endosome rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine and naturally occurring polymers such as polyglucosamine have all been applied to nucleic acid delivery, and are suitable as cationic substances applicable to some aspects of this article. In addition, some researchers have synthesized polymeric substances specifically for nucleic acid delivery. Especially, poly (P-amino ester) is widely used in nucleic acid delivery due to its easy synthesis and biodegradability. In some aspects, this type of synthetic substance can be suitable as cationic substances herein.

As used herein, "polymeric substance" has its common meaning, for example, a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. In some aspects, such repeating units may be all the same; or, in some cases, there are more than one type of repeating units in the polymeric substance. In some cases, the polymeric substance is biologically derived, for example, a biopolymer, such as a protein. In some cases, there may also be additional parts in the polymeric substance, for example, a targeting moiety, such as a targeting moiety described herein.

It will be appreciated by those skilled in the art that when more than one type of repeating unit is present in a polymer (or polymeric portion), the polymer (or polymeric portion) is referred to as a "copolymer". In some aspects, the polymer (or polymeric portion) utilized in accordance with the present disclosure may be a copolymer. The repeating units forming the copolymer may be arranged in any manner. For example, in some aspects, the repeating units may be arranged in a random order; or, or in addition, in some aspects, the repeating units may be arranged in an alternating order, or arranged as a "block" copolymer, which, for example, comprises one or more regions each comprising a first repeating unit (e.g., a first block) and one or more regions each comprising a second repeating unit (e.g., a second block). Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.

In some aspects, the polymeric substance used according to the present disclosure is biocompatible. Biocompatible substances are substances that do not usually cause significant cell death at moderate concentrations. In some aspects, biocompatible substances are biodegradable, for example, can be degraded chemically and/or biologically in a physiological environment, such as in the body. In some aspects, the polymeric substance can be or include protamine or polyalkylene imine, especially protamine.

As will be appreciated by those skilled in the art, 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, inter alia, in association with DNA in place of somatic histones in sperm cells of various animals (e.g., fish). In particular, the term "protamine" is often used to refer to a protein present in fish sperm that is strongly basic, soluble in water, does not coagulate upon heating, and produces primarily arginine upon hydrolysis. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

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

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

Cationic substances (e.g., polymeric substances, including polycationic polymers) contemplated for use herein include those cationic substances capable of electrostatically binding nucleic acids. In some aspects, cationic polymeric substances contemplated for use herein include any cationic polymeric substance that can associate with nucleic acids, for example, by forming a complex with nucleic acids or forming a vesicle that encloses or encapsulates nucleic acids therein.

In some aspects, the particles described herein can include polymers other than cationic polymers, such as non-cationic polymeric species and/or anionic polymeric species. Anionic and neutral polymeric species are collectively referred to herein as non-cationic polymeric species.

B. Lipids and lipid-like substances

The terms "lipid" and "lipidoid material" are used herein to refer to molecules that contain one or more hydrophobic parts or groups and, optionally, one or more hydrophilic parts or groups. According to the present disclosure, lipids and lipidoid materials can be cationic, anionic or neutral. At a selected pH, neutral lipids or lipidoid materials exist in an uncharged or neutral zwitterionic form.

The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides, sterol lipids, and sterol-containing metabolites (such as cholesterol) and prenol lipids. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycoglycerols and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, ceramide sphingomyelin (e.g., sphingomyelin, phosphorylcholine) and glycosphingolipids (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 "lipidoid substance", "lipidoid compound" or "lipidoid 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 are capable of forming an amphiphilic layer when they are present in a vesicle, multilamellar/unilamellar liposome or membrane in an aqueous environment, and includes surfactants or synthetic compounds with hydrophilic and hydrophobic parts. In general, the term refers to molecules that contain hydrophilic and hydrophobic parts with different structural organizations, which may or may not be similar to the hydrophilic and hydrophobic parts of lipids.

In some aspects, RNA solution and lipid formulation mixture thereof or composition can comprise cationic lipid, neutral lipid, cholesterol and/or polymer (such as polyethylene glycol) in conjunction with lipid, and it forms the lipid nanoparticle comprising RNA molecule.Therefore, in some aspects, LNP can comprise cationic lipid and one or more excipients, such as one or more neutral lipid, charged lipid, steroid or steroid analog (such as cholesterol), polymer conjugated lipid (such as PEG-lipid) or its combination.In some aspects, LNP comprises or encapsulates nucleic acid molecule.

i. Cationic lipids

Cation or cationic ionizable lipid or lipidoid material refers to a lipid or lipidoid material that can be positively charged and can electrostatically bind nucleic acid. As used herein, "cationic lipid" or "cationic lipidoid material" refers to a lipid or lipidoid material with a net positive charge. Cationic lipid or lipidoid material binds negatively charged nucleic acid through electrostatic interaction. Generally speaking, cationic lipid has a lipophilic part, such as sterol, acyl chain, diacyl or more acyl chains, and the head group of lipid carries a positive charge usually. Exemplary cationic lipids include one or more amino groups that carry a positive charge. Cationic lipids can encapsulate negatively charged RNA.

In some aspects, cationic lipids are ionizable, so that depending on pH, they can exist in positively charged or neutral forms. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, it is believed that this ionizable behavior enhances efficacy by helping endosome escape and reducing toxicity compared to particles that maintain cationicity at physiological pH. For purposes of this disclosure, unless otherwise contradictory to the situation, the term "cationic lipid" or "cationic lipidoid material" encompasses such "cationic ionizable" lipids or lipidoid materials.

In some aspects, the cationic lipid may account for 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 aspects, the cationic lipid may be at least, at most, just below, or any amount between 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 derived therefrom of the total lipid present in the particle.

Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), dimethyldioctadecyl ammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyl dimethyl ammonium chloride (DODA C), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylammonium (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-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-l-propylammonium trifluoroacetate (DOSPA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (D LenDMA), dioctadecyl amidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybut-4-oxy)-l-(cis, cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentyloxy)-3-dimethyl-l-(cis, cis-9',12'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dialinolenoyloxy-N,N-dimethylpropylamine (DLinDMA), AP), 1,2-N,N'-dilinoleylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dialinolenoyloxycarbamoyl-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), heptathriacont ... A), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylammonium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propylammonium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propylammonium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylammonium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylammonium bromide (bAE -DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-ammonium (DOBAQ), 2-({8-[(3b)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amino (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[bis(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1 ,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-ammonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-ammonium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azadialkyl) 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-l-yl)-9-((4- (dimethylaminobutyryl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propanamide (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipid 02-200); or 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (SM-102).

In some aspects, the lipid nanoparticles comprise one or more cationic lipids. In one aspect, the lipid nanoparticles comprise (4-hydroxybutyl) azadialkyl) bis (hexane-6,1-diyl) bis (2-hexyldecanoate) (ALC-0315), which has the formula:

Cationic lipids are disclosed in, for example, 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 aspects, the RNA-LNP comprises 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. If more than one cationic lipid is incorporated into the LNP, such percentages apply to the combined cationic lipids. In one aspect, the cationic lipid is present in the LNP in an amount such as at least, at most, just below, or between any two thereof, respectively: 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 disclosure, the LNP comprises a combination or mixture of any of the lipids described above.

ii. Polymer-conjugated lipids

In some aspects, LNP comprises polymer-conjugated lipid.Term " polymer-conjugated lipid " refers to the molecule that comprises lipid part and polymer part.The example of polymer-conjugated lipid is pegylated lipid.Term " pegylated lipid " refers to the molecule that comprises lipid part and polyethylene glycol part.Pegylated lipid is known in the art, and comprises 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-s-DMG), 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide etc.

In certain aspects, LNP comprises additional stabilizing lipids, which are polyethylene glycol lipids (PEGylated lipids). Polymer conjugated lipids (e.g., PEG-lipids) refer to molecules comprising both lipid moieties and polymer moieties. Examples of polymer conjugated lipids are PEG-lipids. PEG-lipids refer to molecules comprising both lipid moieties and polyethylene glycol moieties. PEG-lipids include, but are not limited to, 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. Representative polyethylene glycol lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one aspect, the polyethylene glycol lipid is N-[(methoxypolyethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxypropane-3-amino (PEG-c-DMA). In one aspect, the polyethylene glycol lipid is PEG-2000-DMG. In one aspect, the polyethylene glycol lipid is PEG-c-DOMG. In other aspects, the LNP comprises polyethylene glycol diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), polyethylene glycol phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate (PEG-S- In some embodiments, the present invention relates to PEG-lipids, such as poly(DMG), poly(ethylene glycol) ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(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 aspects, the lipid nanoparticle comprises a polymer-conjugated lipid. In one aspect, the lipid nanoparticle comprises 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159), which has the formula:

In various aspects, the molar ratio of the cationic lipid to the PEGylated lipid is in the range of about 100: 1 to about 20: 1, for example, 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 derived therein.

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

iii. Additional lipids

In certain aspects, LNP comprises one or more additional lipids or lipidoid materials, which stabilize the formation of particles during particle formation. Suitable stabilization or structural lipids include non-cationic lipids, such as neutral lipids and anionic lipids. Without being bound by any theory, the formulation optimization of LNP can enhance particle stability and nucleic acid delivery effectiveness by adding other hydrophobic moieties, such as cholesterol and lipids other than ionizable/cationic lipids or lipidoid materials.

As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. In some aspects, 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 a phospholipid and cholesterol or a derivative thereof.

Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin and cerebroside. Exemplary phospholipids include, for example, phosphatidylcholine, for example, diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), diarachidonylphosphatidylcholine (DAPC), dibehenylphosphatidylcholine (DMPC), di- ...-behenylphosphatidylcholine (DMPC), di-pentadecanoylphosphatidylcholine, di-pentadecanoylphosphatidylcholine, choline (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), l-oleoyl-2-cholesterol hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), and 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC); and phosphatidylethanolamines, for example, diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), diphytanoylphosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one aspect, the neutral lipid is 1,2-distearoyl-sn-glycero-3 phosphocholine (DSPC), which has the formula:

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

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

In certain aspects, 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 aspect, cholesterol has the formula:

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 charge, particle size, stability, tissue selectivity and biological activity of the nucleic acid. Thus, in some aspects, 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 aspects, non-cationic lipids, such as neutral lipids (e.g., one or more phospholipids and/or cholesterol) can account for 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 lipids present in the particle. In some aspects, non-cationic lipids, such as neutral lipids (e.g., one or more phospholipids and/or cholesterol) can be at least, at most, just the following amount, or any amount between them: 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol% of the total lipids present in the particle.

VI. Characterization and Analysis of RNA Molecules

The RNA molecules described herein can be analyzed and characterized using various methods. Can be analyzed before or after capping. Alternatively, can be analyzed before or after affinity purification based on poly A capture. In another aspect, can be analyzed before or after additional purification steps, such as anion exchange chromatography and similar steps thereof. For example, an electrophoresis system based on a bioanalyzer chip can be used to determine RNA template quality. In other aspects, analytical reverse phase HPLC is used to analyze RNA template purity respectively. Capping efficiency can be analyzed using, for example, total nuclease digestion, followed by MS/MS quantification of dinucleotide end cap species relative to uncapped GTP species. In vitro efficacy can be analyzed by, for example, transfecting RNA molecules into human cell lines. Methods such as ELISA or flow cytometry can be used to quantify the protein expression of the polypeptide of interest. Immunogenicity can be analyzed by, for example, transfecting RNA molecules into cell lines (e.g., PBMC) indicating innate immune stimulation. Cytokine induction can be analyzed using, for example, methods such as ELISA, thereby quantifying cytokines, such as interferon-α. Biodistribution can be analyzed, for example, by bioluminescence measurement.

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

In some aspects, references include organisms that administer RNA polynucleotides that are otherwise similar but do not have the m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2 end caps. In some aspects, references include organisms that administer RNA polynucleotides that are otherwise similar but do not have the end cap proximal sequences disclosed herein. In some aspects, references include organisms that administer RNA polynucleotides that are otherwise similar but have self-hybridizing sequences.

In some aspects, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured. In some aspects, at least 24 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured. In some aspects, at least 48 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured. In some aspects, at least 72 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured. In some aspects, at least 96 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured. In some aspects, at least 120 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used, the expression of the increase is measured.

In some aspects, the expression of elevation is measured at about 24-120 hours after the composition or pharmaceutical preparation comprising RNA polynucleotides is used. In some aspects, the expression of elevation is measured at 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 the composition or pharmaceutical preparation comprising RNA polynucleotides is used.

In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 2 times to at least 10 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 2 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 3 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 4 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 6 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 8 times. In some aspects, the expression of the gene (e.g., antigen) of interest is increased by at least 10 times.

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

In some aspects, the expression of a gene (such as an antigen) of interest is increased (such as expression duration increases) after applying a composition or pharmaceutical preparation comprising RNA polynucleotides for at least, at most, just the following time or any time between the two: 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours. In some aspects, the expression of a gene (such as an antigen) of interest is increased after application for at least 24 hours. In some aspects, the expression of a gene (such as an antigen) of interest is increased after application for at least 48 hours. In some aspects, the expression of a gene (such as an antigen) of interest is increased after application for at least 72 hours. In some aspects, the expression of a gene (such as an antigen) of interest is increased after application for at least 96 hours. In some aspects, the expression of a gene (such as an antigen) of interest is increased after application for at least 120 hours.

In some aspects, the expression of paid attention to gene (such as antigen) rises and continues about 24-120 hours after using the composition or pharmaceutical preparation comprising RNA polynucleotide.In some aspects, expression rises and continues 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 using the composition or pharmaceutical preparation comprising RNA polynucleotide. In some aspects, the elevated expression of a gene of interest (e.g., an antigen) persists for at least, at most, exactly, or any time between 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or any range or value derivable therein.

VII. Immune Response and Analysis

As discussed herein, the present disclosure relates to inducing or eliciting an immune response in a subject against a VZV protein (e.g., a wild-type or variant VZV glycoprotein). In one aspect, the immune response can protect a subject from developing an infection or related disease (particularly those associated with VZV) or treat a subject who has, is suspected of having, or is at risk of developing the infection or related disease. One use of the immunogenic compositions of the present disclosure is to prevent VZV infection by inoculating or vaccinating a subject.

A. Immunoassay

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. Many types of immunoassays 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 and immunocytochemistry of labeled ligands in vitro and in vivo.

Immunoassay is generally a binding assay. In some aspects, immunoassay is various types of enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly suitable. In one example, the antibody or antigen is fixed on a selected surface, such as a hole in a polystyrene microtiter plate, a test paper or a column carrier. Then, a test composition (such as a clinical sample) suspected of containing the desired antigen or antibody is added to the hole. After combining and washing to remove the immune complex of non-specific binding, the combined antigen or antibody can be detected. Detection is generally achieved by adding another antibody that is specific to the desired antigen or antibody that is connected to a detectable label. This type of ELISA is referred to as a "sandwich ELISA". Detection can also be achieved by adding a second antibody that is specific to the desired antigen, followed by adding a third antibody that has binding affinity to the second antibody, wherein the third antibody is connected to a detectable label.

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

Antigen or antibody can also be connected to a solid support, such as a solid support in the form of a disc, bead, test paper, membrane or column matrix, and the sample to be analyzed is applied to the fixed antigen or antibody. When the plate is coated with antigen or antibody, the wells of the culture plate are generally incubated overnight or for a specified time with the antigen or antibody solution. The wells of the culture plate are then washed to remove incompletely adsorbed substances. Then, any residual available surface in the hole is "coated" with a nonspecific protein that is antigenically neutral to the test antiserum. Proteins include bovine serum albumin (BSA), casein and milk powder solution. The coating allows blocking of nonspecific adsorption sites on the fixed surface and therefore reduces the background caused by the nonspecific binding of antiserum on the surface.

B. Diagnosis of VZV infection

The present disclosure encompasses 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, methods of detecting the presence of infection involve the step of obtaining a sample suspected of being infected with one or more strains of VZV, such as a sample obtained from a subject, for example, a sample obtained from blood, saliva, tissue, bone, muscle, cartilage or skin. After separation 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 analytical techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassay, Western blot analysis, and ELISA analysis.

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

Thus, RNA molecules encoding VZV polypeptides, proteins and/or peptides according to the present disclosure can be used to treat, prevent or reduce the severity of disease caused by infection with VZV (eg, active or passive immunization) or as research tools.

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

C. Protective immunity

In some 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 specific disease or condition to which an agent against which an immune response exists. An immunogenic effective amount is capable of conferring protective immunity to a subject.

As used herein, the phrase "immune response" or its equivalent phrase "immunological response" refers to the production of a humoral response (antibody-mediated), a cellular response (mediated by antigen-specific T cells or their secretory products), or both a humoral and a cellular response to an antigen. Such responses may be active or passive. A cellular immune response is initiated by the presentation of polypeptide epitopes in association with class I or class II MHC molecules to activate antigen-specific CD4(+) T helper cells and/or CD8(+) cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microglia, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred on a subject by 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 immunity" includes, but is not limited to, the administration of activated immune effectors, including cellular mediators or protein mediators of immune responses (e.g., monoclonal and/or polyclonal antibodies). Monoclonal or polyclonal antibody compositions can be used for passive immunization to treat, prevent, or reduce the severity of diseases caused by infection with organisms carrying antigens recognized by antibodies. Antibody compositions may include antibodies that bind to a variety of antigens, which may then associate with various organisms. The antibody component may be a polyclonal antiserum. In certain aspects, one or more antibodies are affinity purified from an animal or a second subject that has been challenged with an antigen. Alternatively, an antibody mixture may be used, which is a mixture of monoclonal and/or polyclonal antibodies against antigens present in the same, related or different microorganisms or organisms (such as viruses, including but not limited to VZV).

Passive immunity can be conferred to a patient or subject by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from a donor or other non-patient source of known immunoreactivity. In other aspects, an immunogenic composition of the disclosure can be administered to a subject, who then serves as a source or donor of globulins containing antibodies to VZV or other organisms, which are produced in response to challenge with the immunogenic composition ("hyperimmune globulins"). The subject thus treated will be provided with plasma, from which hyperimmune globulins are then obtained via conventional plasma separation methods and administered to another subject to confer resistance to or treat VZV infection.

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

The presence of a cell-mediated immune response can be determined by proliferation assays (CD4(+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contributions of humoral and cellular responses to the protective or therapeutic effects of an immunogenic composition can be distinguished by isolating IgG and T cells, respectively, from immunized isogenic animals and measuring the protective or therapeutic effects 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 immune response in an animal or recipient, including IgG, IgD, IgE, IgA, IgM, and related proteins. Under normal physiological conditions, antibodies are present in plasma and other body fluids, as well as in the membranes of certain cells, and are produced by lymphocytes of a type designated as B cells, or their functional equivalents.

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

VIII. Composition

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

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

Preservatives suitable for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, the term "excipient" refers to a substance that may be present in 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, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

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

The term "carrier" refers to a component that can be natural, synthetic, organic, or inorganic, in which the active ingredient is incorporated to facilitate, enhance, or achieve the administration of the pharmaceutical composition. As used herein, carriers can be one or more compatible solid or liquid fillers, diluents, or encapsulated materials suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxypropylene copolymers. In some aspects, the pharmaceutical composition of the present disclosure includes 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).

The selection of the pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In some aspects, the composition comprises an RNA molecule containing an open reading frame encoding an immunogenic polypeptide. In some aspects, the immunogenic polypeptide comprises a VZV antigen. In some aspects, the VZV antigen is a VZV polypeptide. In some aspects, 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 aspects, 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 VZVgL polypeptide, the RNA molecule encodes a VZV gI polypeptide, and/or the RNA molecule encodes a VZV gE polypeptide. In one aspect, the RNA molecule encodes a VZV gE polypeptide. In some aspects, the VZV polypeptide comprises two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, or more) VZV polypeptides.

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

A. Immunogenic Compositions Including LNPs

In some aspects, the pharmaceutical composition comprises an RNA molecule (e.g., polynucleotide) disclosed herein formulated with a lipid-based delivery system. Therefore, in some aspects, the composition includes a lipid-based delivery system (e.g., LNP) (e.g., a lipid-based vaccine), which delivers nucleic acid molecules to the interior of the cell, which can then replicate, inhibit protein expression, and/or express the encoded polypeptide of interest within the cell. The delivery system may have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some aspects, the composition comprises at least one RNA molecule encoding a VZV polypeptide, which is compounded with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids, and forms lipid nanoparticles (LNPs), liposomes, lipid complexes, and/or nanoliposomes. In some aspects, the composition comprises lipid nanoparticles. Therefore, in certain aspects, the disclosure relates to a composition comprising one or more lipids (e.g., VZV RNA-LNPs) associated with nucleic acids or polypeptides/peptides.

In some cases, the immunogenic composition comprising a lipid-based delivery system may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents and/or excipients. In some respects, the immunogenic composition comprising a lipid-based delivery system further includes a pharmaceutically acceptable vehicle. In some respects, each of a buffer, a stabilizer and an optional salt may be included in the immunogenic composition comprising a lipid-based delivery system. In other respects, any one or more of a buffer, a stabilizer, a salt, a surfactant, a preservative and an excipient may not be included in the immunogenic composition comprising a lipid-based delivery system.

In another aspect, the immunogenic composition including a lipid-based delivery system further comprises a stabilizer. In some aspects, the stabilizer comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, polydextrose, polyvinylpyrrolidone, glycine or a combination thereof. In some aspects, the stabilizer is a disaccharide or sugar. In one aspect, the stabilizer is sucrose. In another aspect, the stabilizer is trehalose. In another aspect, the stabilizer is a combination of sucrose and trehalose. In some aspects, the total concentration of the stabilizer in the composition is about 5% to about 10% w/v. For example, the total concentration of the stabilizer may be at least, at most, exactly equal to, or between any two of the following: 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 derived therefrom. In some aspects, the stabilizer 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 aspects, the concentration of the stabilizer is at least, at most, exactly equal to, or between any two of 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, or more.

In another aspect, the amount of the mass of the stabilizer is in a specific ratio to the amount of the mass of the RNA. In one aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 5000. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 2000. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 1000. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 500. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 100. In another aspect, the ratio of the amount of the mass of the stabilizer to the pharmaceutical substance is no more than 50. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 10. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 1. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 0.5. In another aspect, the ratio of the amount of the mass of the stabilizer to the RNA is no more than 0.1. In another aspect, the stabilizer and the RNA comprise a mass ratio of about 200 to 2000:1 of the stabilizer and the RNA.

In some aspects, the immunogenic composition comprising a lipid-based delivery system further comprises a buffer. Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, Tris hydrochloric acid (HCl), sulfamate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol and/or a combination thereof. In some aspects, the buffer is a HEPES buffer, a Tris buffer or a PBS buffer. In one aspect, the buffer is a Tris buffer. In another aspect, the buffer is a HEPES buffer. In another aspect, the buffer is a PBS buffer. For example, the buffer concentration may be at least, at most, just equal to or between any two of the following: 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 may be derived from any range or value therein. The buffer may be neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0 or pH 7.2 to pH 7.6. For example, the buffer can be at least, at most, just below pH, or any of between 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In a specific aspect, the buffer is at pH 7.4.

In some aspects, the immunogenic composition comprising a 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 aspect, the salt is a sodium salt. In a specific aspect, the sodium salt is sodium chloride. In one aspect, the salt is a potassium salt. In some aspects, the potassium salt comprises potassium chloride. The concentration of the salt in the composition may be from about 70mM to about 140mM. For example, the salt concentration may be at least, at most, just equal to, or between any two of the following: 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, or 200mM.

In some aspects, 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 aspects, the concentration of the salt is at least, at most, just equal to, or between any two of the following: 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 more. The salt may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the salt may be at a pH of at least, at most, exactly, 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 aspects, the immunogenic composition comprising a lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof. As used herein, "any other excipient" includes, but is not limited to, antioxidants, glutathione, EDTA, methionine, desferal methanesulfonate, antioxidants, metal scavengers, or free radical scavengers. In one aspect, 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, divalent cations, lactated Ringer's solution, amino acids, sugars, polyols, polymers, or cyclodextrins.

Excipients refer to ingredients in the immunogenic composition that are not active ingredients, examples of which include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or coloring agents. Preservatives used in the compositions disclosed herein include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal. As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcohol/water solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), 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, flavoring agents, dyes, fluids, and nutritional supplements, such as those known to those of ordinary 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%, from about 25% to about 75%, from about 30% to about 60%, or from about 12% to about 60% by weight of the total composition.

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

In one aspect, the pharmaceutical composition comprises a VZV RNA molecule encoding a VZV polypeptide as disclosed herein, which is 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 aspects, the VZV RNA-LNP composition is a liquid. In some aspects, the VZV RNA-LNP composition is frozen. In some aspects, the VZV RNA-LNP composition is lyophilized. In some aspects, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide molecule encoding a VZV polypeptide as disclosed herein, which is encapsulated in a LNP composed of a lipid having a cationic lipid, a pegylated lipid (i.e., a PEG-lipid) and one or more structural lipids (e.g., a neutral lipid).

In some aspects, the VZV RNA-LNP composition comprises a cationic lipid. The cationic lipid may comprise any one or more cationic lipids disclosed herein. In a specific aspect, the cationic lipid comprises ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315). In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.6 6, 0.67, 0.6 8. 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, 1.2 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, 1.98, 1.99 or 2 ng/μg/mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least, at most, between any two of, or just below 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, 0.70, 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 4, 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. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least 0.4 mg/mL, at least 0.45 mg/mL, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.5, between 0.5 and 0.6, between 0.6 and 0.7, between 0.7 and 0.8, between 0.8 and 0.9, or between 0.9 and 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL, between 0.5 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL.

In a particular aspect, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8 to 0.95 mg/mL. In a particular aspect, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.8 to 0.9 mg/mL. In a particular aspect, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.85 to 0.9 mg/mL. In a particular aspect, a 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 particular aspect, a cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of about 0.86 mg/mL. The concentration of the lyophilized composition was determined after reconstitution.

In some aspects, the VZV RNA-LNP composition further comprises a PEGylated lipid (ie, a PEG-lipid). The PEGylated lipid may comprise any one or more of the PEGylated lipids disclosed herein. In a specific aspect, the PEGylated lipid comprises 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159). In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 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, 1. 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/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 , 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.5 mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least 0.01 mg/mL, at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, 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 aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of between 0.01 and 0.05 mg/mL, between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, or between 0.2 and 0.25 mg/mL.

In a particular aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.05 to 0.15 mg/mL. In a particular aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.10 to 0.15 mg/mL. In a particular aspect, 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 aspect, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of about 0.11 mg/mL. The concentration of the lyophilized composition is measured after reconstitution.

In some aspects, the VZV RNA-LNP composition further comprises one or more structural lipids. The one or more structural lipids may comprise any one or more structural lipids disclosed herein. In a specific aspect, the one or more structural lipids comprise a neutral lipid and a steroid or a steroid analog. In a specific aspect, the one or more structural lipids comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.4 .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, 0.9 2, 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, 1.4 6, 1.47, 1.48 ,1.49,1.5,1.51,1.52,1.53,1.54,1.55,1.56,1.57,1.58,1.59,1.6,1.61,1.62,1.63,1.64,1.65,1.66,1.67,1.68,1.69,1.7,1.71,1.72,1.7 3.1.74.1.75 , 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/mL. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 10. 44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, 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, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, one or more structural lipids (e.g., DSPC and cholesterol) are present at between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL. Concentrations between 0.5 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL are included in the composition.

In a particular aspect, one or more structural lipids include DSPC, and DSPC is included in the composition with a concentration of about 0.1 to 0.25mg/mL. In a particular aspect, one or more structural lipids include DSPC, and DSPC is included in the composition with a concentration of about 0.15 to 0.25mg/mL. In a particular aspect, one or more structural lipids include DSPC, and DSPC is included in the composition with 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 0.25mg/mL. In a particular aspect, DSPC is included in the composition with a concentration of about 0.19mg/mL.

In a particular aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of about 0.3 to 0.45mg/mL. In a particular aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of about 0.3 to 0.4mg/mL. In a particular aspect, one or more structural lipids include cholesterol, and cholesterol is included in the composition at a concentration of about 0.35 to 0.45mg/mL. In a particular aspect, one or more structural lipids include cholesterol, and 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 0.45mg/mL. In a particular aspect, cholesterol is included in the composition at a concentration of about 0.37mg/mL. The concentration of the lyophilized composition is measured after reconstitution.

In some aspects, the VZV RNA-LNP composition further comprises one or more buffers and stabilizers and optionally a salt diluent. Thus, in some aspects, the VZV RNA-LNP composition comprises a cationic lipid, a pegylated lipid, one or more structural lipids, one or more buffers, a stabilizer and optionally a salt diluent.

In some aspects, the VZV RNA-LNP composition comprises one or more buffers. The one or more buffers may comprise any one or more buffers disclosed herein. In a specific aspect, the composition comprises a Tris buffer comprising at least a first buffer and a second buffer. In some aspects, the first buffer is tromethamine. In some aspects, the second buffer is Tris hydrochloric acid (HCl). In some aspects, the first buffer and the second buffer of the Tris buffer (e.g., tromethamine and Tris HCl) is included in the composition at a concentration of at least, at most, between any two of, or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.6 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, 1.21, 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, 1.4 8, 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/mL. The concentration of the lyophilized composition is determined after reconstitution.

In some aspects, the VZV RNA-LNP composition is a liquid composition comprising a Tris buffer. In some aspects, the Tris buffer comprises a first buffer. In some aspects, the first buffer is tromethamine. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of at least, at most, between any two of, or just below: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 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 .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 aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of at least 0.1 mg/mL, at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, 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, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, the first buffer (e.g., tromethamine) is included in the liquid composition at a concentration of between 0.05 and 0.15, between 0.15 and 0.25, between 0.25 and 0.35, between 0.35 and 0.45, between 0.45 and 0.55, between 0.55 and 0.65, between 0.65 and 0.75, between 0.75 and 0.85, or between 0.85 and 0.95 mg/mL. In some aspects, the first buffer (e.g., tromethamine) is present at between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL, between 0.5 Concentrations of between 0.1 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL are included in the liquid composition.

In a particular aspect, the first buffer (such as tromethamine) is included in the liquid composition with a concentration of about 0.1 to 0.3mg/mL. In a particular aspect, the first buffer (such as tromethamine) is included in the liquid composition with a concentration of about 0.15 to 0.25mg/mL. In a particular aspect, the first buffer (such as tromethamine) is included in the liquid composition with 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 0.3mg/mL. In a particular aspect, the first buffer (such as tromethamine) is included in the liquid composition with a concentration of about 0.20mg/mL.

In some aspects, the VZV RNA-LNP composition is a liquid composition comprising a Tris buffer containing a second buffer. In some aspects, the second buffer comprises Tris HCl. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of at least, at most, between any two of, or just below: 0.5, 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18 , 1.19, 1.20, 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. In some aspects, the second buffer (e.g., TrisHCl) is included in the liquid composition at a concentration of at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, at least 1 mg/mL, at least 1.05 mg/mL, at least 1.10 mg/mL, at least 1.15 mg/mL, at least 1.20 mg/mL, at least 1.25 mg/mL, at least 1.30 mg/mL, at least 1.35 mg/mL, at least 1.40 mg/mL, at least 1.45 mg/mL, or at least 1.50 mg/mL. In some aspects, a second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration between 0.5 and 0.6 mg/mL, between 0.6 and 0.7 mg/mL, between 0.7 and 0.8 mg/mL, between 0.8 and 0.9 mg/mL, between 0.9 and 1 mg/mL, between 1 and 1.10 mg/mL, between 1.10 and 1.20 mg/mL, between 1.20 and 1.30 mg/mL, between 1.30 and 1.40 mg/mL, or between 1.40 and 1.50 mg/mL.

In a particular aspect, the second buffer (such as Tris HCl) is included in the liquid composition with a concentration of about 1.25 to 1.40mg/mL. In a particular aspect, the second buffer (such as Tris HCl) is included in the liquid composition with a concentration of about 1.30 to 1.40mg/mL. In a particular aspect, the second buffer (such as Tris HCl) is included in the liquid composition with 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 1.40mg/mL. In a particular aspect, the second buffer (such as Tris HCl) is included in the liquid composition with a concentration of about 1.32mg/mL.

In some aspects, the VZV RNA-LNP composition is a lyophilized composition comprising a Tris buffer. In some aspects, the Tris buffer comprises a first buffer. In some aspects, the first buffer is tromethamine. In some aspects, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstituted concentration of at least, at most, between any two of, or just below: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstituted concentration of at least 0.01 mg/mL, at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, 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 aspects, a first buffer (e.g., tromethamine (Tris base)) is included in the lyophilized composition at a reconstituted concentration of between 0.01 and 0.05 mg/mL, between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, or between 0.45 and 0.5 mg/mL.

In a particular aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstitution concentration of about 0.01 and 0.15 mg/mL. In a particular aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstitution concentration of about 0.01 and 0.10 mg/mL. In a particular aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstitution concentration of about 0.05 and 0.15 mg/mL. In a particular aspect, the first buffer (e.g., tromethamine) is included in the lyophilized composition at a reconstitution 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. In certain aspects, the first buffer (eg, tromethamine) is included in the lyophilized composition at a post-reconstitution concentration of about 0.09 mg/mL.

In some aspects, the VZV RNA-LNP composition is a lyophilized composition comprising a Tris buffer containing a second buffer. In some aspects, the second buffer comprises Tris HCl. In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a reconstituted concentration of at least, at most, between any two of, or just below 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, 0.70, 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, 0.86, 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, 0. 94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition with a reconstitution concentration of at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL, at least 0.4 mg/mL, at least 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, or at least 1 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition with a reconstitution concentration between 0.1 and 0.2 mg/mL, between 0.2 and 0.3 mg/mL, between 0.3 and 0.4 mg/mL, between 0.4 and 0.5 mg/mL, between 0.5 and 0.6 mg/mL, between 0.6 and 0.7 mg/mL, between 0.7 and 0.8 mg/mL, between 0.8 and 0.9 mg/mL, or between 0.9 and 1 mg/mL.

In a particular aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a reconstitution concentration of about 0.5 and 0.65 mg/mL. In a particular aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a reconstitution concentration of about 0.5 and 0.6 mg/mL. In a particular aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a reconstitution concentration of about 0.55 and 0.65 mg/mL. In a particular aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a reconstitution 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. In certain aspects, a second buffer (eg, TrisHCl) is included in the lyophilized composition at a post-reconstitution concentration of about 0.57 mg/mL.

In some aspects, the VZV RNA-LNP composition comprises a stabilizer. The stabilizer may comprise any one or more stabilizers disclosed herein. In some aspects, the stabilizer also acts as a cryoprotectant. In specific aspects, the stabilizer comprises sucrose. In some aspects, the stabilizer (e.g., sucrose) is included in the composition at a concentration of at least, at most, between any two of, or just below: 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 9, 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 ,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,1 09, 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, 13 9, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 15 6, 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/mL.

In some aspects, the VZV RNA-LNP composition is a liquid composition, and the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of at least, at most, between any two of, or just below: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115 5, 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 aspects, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of at least 70 mg/mL, at least 75 mg/mL, at least 80 mg/mL, at least 85 mg/mL, at least 90 mg/mL, at least 95 mg/mL, at least 100 mg/mL, at least 105 mg/mL, at least 110 mg/mL, at least 115 mg/mL, at least 120 mg/mL, at least 125 mg/mL, or at least 130 mg/mL. In some aspects, stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of between 70 and 80 mg/mL, between 80 and 90 mg/mL, between 90 and 100 mg/mL, between 100 and 110 mg/mL, between 110 and 120 mg/mL, or between 120 and 130 mg/mL.

In a particular aspect, stabilizer (such as sucrose) is included in the liquid composition with a concentration of about 95 to 110mg/mL. In a particular aspect, stabilizer (such as sucrose) is included in the liquid composition with a concentration of about 95 to 105mg/mL. In a particular aspect, stabilizer (such as sucrose) is included in the liquid composition with a concentration of about 100 to 110mg/mL. In a particular aspect, stabilizer (such as sucrose) is included in the liquid composition with a concentration of about 95,96,97,98,99,100,101,102,103,104,105,106,107,108,109 or 110mg/mL. In a particular aspect, stabilizer (such as sucrose) is included in the liquid composition with a concentration of about 103mg/mL.

In some aspects, the VZV RNA-LNP composition is a lyophilized composition and the stabilizer (e.g., sucrose) is included in the lyophilized composition at a post-reconstitution concentration of at least, at most, between any two of, or just below 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 aspects, stabilizers (e.g., sucrose) are included in the lyophilized composition at a reconstitution concentration of at least 20 mg/mL, at least 25 mg/mL, at least 30 mg/mL, at least 35 mg/mL, at least 40 mg/mL, at least 45 mg/mL, at least 50 mg/mL, at least 55 mg/mL, at least 60 mg/mL, at least 65 mg/mL, at least 70 mg/mL, at least 75 mg/mL, or at least 80 mg/mL. In some aspects, stabilizers (e.g., sucrose) are included in the lyophilized composition at a reconstitution concentration of between 20 and 30 mg/mL, between 30 and 40 mg/mL, between 40 and 50 mg/mL, between 50 and 60 mg/mL, between 60 and 70 mg/mL, or between 70 and 80 mg/mL.

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

In some aspects, the lyophilized composition is reconstituted in a suitable carrier or diluent. The carrier or diluent may comprise any one or more of the carriers or diluents disclosed herein. In a particular aspect, the carrier or diluent comprises a salt diluent such as sodium chloride (NaCl) (e.g., saline, such as normal saline or standard saline). The sodium chloride may comprise 0.9% sodium chloride injection. In some aspects, the lyophilized composition is reconstituted in saline in at least the following amounts, at most the following amounts, any amount between the following, or exactly the following amounts: 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. In some aspects, the lyophilized composition is reconstituted in at least 0.1 mL, at least 0.2 mL, at least 0.3 mL, at least 0.4 mL, at least 0.5 mL, at least 0.6 mL, at least 0.7 mL, at least 0.8 mL, at least 0.9 mL, or at least 1 mL of sodium chloride.

In a particular aspect, the lyophilized composition is reconstituted in about 0.6 to 0.75 mL of sodium chloride/saline. In a particular aspect, the lyophilized composition is reconstituted in about 0.65 to 0.75 mL of sodium chloride/saline. In a particular aspect, 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.

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, 27.5, 27.5, 28, 28.5, 29, 30.5, 31, 32.5, 33, 34, 35.5, 36, 37.5, 38, 39, 40.5, 41, 42.5, 43, 44, 45.5, 46, 47.5, 48, 49, 50.5, 51 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50 ng/μg/mg/mL. In some aspects, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration of at least, at most, between any two of, or just below 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 aspects, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration of at least 1 mg/mL, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 6 mg/mL, at least 7 mg/mL, at least 8 mg/mL, at least 9 mg/mL, at least 10 mg/mL, at least 11 mg/mL, at least 12 mg/mL, at least 13 mg/mL, at least 14 mg/mL, at least 15 mg/mL, or at least 20 mg/mL.

In a particular aspect, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a reconstitution concentration of about 5 and 15 mg/mL. In some aspects, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a reconstitution concentration between about 5 and 10 mg/mL. In a particular aspect, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a reconstitution concentration of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL. In a particular aspect, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a reconstitution concentration of about 9 mg/mL.

The pH of the VZV RNA-LNP composition can be at least, at most, just below, or between any two of the following: pH 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derived therefrom. In some aspects, the VZV RNA-LNP composition is at a pH of at least 6.5, at least 7.0, at least 7.5, at least 8.0, or at least 8.5. In specific aspects, the VZV RNA-LNP composition is at a pH between 6.0 and 7.5, between 6.5 and 7.5, between 7.0 and 8.0, between 7.5 and 8.5. In specific aspects, the VZV RNA-LNP composition is between 7.0 and 8.0. In certain aspects, the VZV RNA-LNP composition is at 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 certain aspects, the VZV RNA-LNP composition is at about pH 7.4. In some aspects, sodium hydroxide buffer can be used for buffer pH adjustment.

In a specific aspect, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, encapsulated in an LNP having the following lipid composition: 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL; and a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL.

In a particular aspect, the VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, encapsulated in an LNP having the following lipid composition: ALC-0315 at a concentration of about 0.8 to 0.95 mg/mL; ALC-0159 at a concentration of about 0.05 to 0.15 mg/mL; 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.

In a specific aspect, 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 to 0.3 mg/mL, a second buffer at a concentration of about 1.25 to 1.4 mg/mL, and a stabilizer at a concentration of about 95 to 110 mg/mL. In a specific aspect, 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 to 0.3 mg/mL, Tris HCl at a concentration of about 1.25 to 1.4 mg/mL, and sucrose at a concentration of about 95 to 110 mg/mL.

Thus, in a particular aspect, the liquid VZV RNA-LNP composition comprises 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL, a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL, and further comprises a first buffer at a concentration of about 0.1 to 0.3 mg/mL, a second buffer at a concentration of about 1.25 to 1.4 mg/mL, and a stabilizer at a concentration of about 95 to 110 mg/mL.

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

In a specific aspect, 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 buffer at a concentration of about 0.01 and 0.15 mg/mL, a second buffer at a concentration of about 0.5 and 0.65 mg/mL, a stabilizer at a concentration of about 35 to 50 mg/mL, and a salt diluent at a concentration between about 5 and 15 mg/mL.

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

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

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

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

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

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

The VZV RNA-LNP composition further comprises a VZV RNA described herein encapsulated in the LNP, see Section D. Administration.

In a particular aspect, the VZV RNA-LNP composition is a liquid VZV RNA-LNP composition comprising a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, the VZV RNA polynucleotide having a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, the VZV RNA polynucleotide being encapsulated in a LNP having the following lipid composition: 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL; and the liquid VZV The RNA-LNP composition further comprises a buffer composition comprising a first buffer at a concentration of about 0.15 to 0.3 mg/mL, a second buffer at a concentration of about 1.25 to 1.4 mg/mL, and a stabilizer at a concentration of about 95 to 110 mg/mL.

In a specific aspect, the liquid VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, the VZV RNA polynucleotide having a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, and more preferably about 0.06 mg/mL, the VZV RNA polynucleotide being encapsulated in a LNP having the following lipid composition: ALC-0315 at a concentration of about 0.8 to 0.95 mg/mL, ALC-0159 at a concentration of about 0.05 to 0.15 mg/mL, 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; and the liquid VZV The RNA-LNP composition further comprises a Tris buffer composition comprising: tromethamine at a concentration of about 0.1 to 0.3 mg/mL, TrisHCl at a concentration of about 1.25 to 1.4 mg/mL, and sucrose at a concentration of about 95 to 110 mg/mL.

In a specific aspect, the VZV RNA-LNP composition is a lyophilized VZV RNA-LNP composition comprising a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, the VZV RNA polynucleotide having a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, the VZV RNA polynucleotide being encapsulated in a LNP having the following lipid composition: 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 first structural lipid at a concentration of about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of about 0.3 to 0.45 mg/mL; and the lyophilized VZV The RNA-LNP composition further comprises a first buffer at a concentration of about 0.01 and 0.15 mg/mL, a second buffer at a concentration of about 0.5 and 0.65 mg/mL, a stabilizer at a concentration of about 35 to 50 mg/mL, and a salt diluent at a concentration of 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of the salt diluent. The concentration in the lyophilized VZV RNA-LNP composition is determined after reconstitution.

In a specific aspect, the lyophilized VZV RNA-LNP composition comprises a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, the VZV RNA polynucleotide having a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, and more preferably about 0.06 mg/mL, the VZV RNA polynucleotide being encapsulated in a LNP having the following lipid composition: ALC-0315 at a concentration of about 0.8 to 0.95 mg/mL, ALC-0159 at a concentration of about 0.05 to 0.15 mg/mL, 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; and the lyophilized VZV The RNA-LNP composition further comprises tromethamine at a concentration of about 0.01 and 0.15 mg/mL, Tris HCl at a concentration of about 0.5 and 0.65 mg/mL, sucrose at a concentration of about 35 to 50 mg/mL, and NaCl at a concentration of about 5 to 15 mg/mL. In a specific aspect, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of NaCl diluent (saline). The concentration in the lyophilized VZV RNA-LNP composition is determined after reconstitution.

In some aspects, the VZV RNA-LNP composition (before lyophilization) comprises a VZV RNA polynucleotide encoding a VZV polypeptide as disclosed herein, the VZV RNA polynucleotide having a concentration of at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, the VZV RNA polynucleotide being encapsulated in a LNP having the following lipid composition: a cationic lipid at a concentration of about 1.0 to 3.0 mg/mL, a PEGylated lipid at a concentration of about 0.10 to 0.35 mg/mL, a first structural lipid at a concentration of about 0.4 to 0.55 mg/mL, a second structural lipid at a concentration of about 0.85 to 1.0 mg/mL; and the VZV The RNA-LNP composition (before lyophilization) further comprises a first buffer at a concentration of about 0.1 and 0.3 mg/mL, a second buffer at a concentration of about 1.25 and 1.40 mg/mL, and a stabilizer at a concentration of about 95 to 110 mg/mL.

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

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

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

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

In some aspects, the RNA-LNP immunogenic composition (before lyophilization) comprises an RNA molecule/polynucleotide encoding a VZV polypeptide as disclosed herein, wherein the concentration of the RNA molecule/polynucleotide is at least, at most, exactly 0.01, 0.15, 0.30, 0.45, 0.60, 0.75 or 0.90 mg/mL or any concentration therebetween, preferably about 0.01 to 0.09 mg/mL, and more preferably 0.15 mg/mL, and the RNA molecule/polynucleotide is encapsulated in LNP; and the RNA-LNP immunogenic composition (before lyophilization) 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. Vaccine

In some aspects, the pharmaceutical composition described herein is an immunogenic composition for inducing an immune response. For example, in some aspects, the immunogenic composition is a vaccine. In some aspects, the composition described herein includes at least one isolated nucleic acid or polypeptide molecule as described herein. In a specific aspect, the immunogenic composition comprises nucleic acid, and the immunogenic composition is a nucleic acid vaccine. In some aspects, the immunogenic composition comprises RNA (e.g., mRNA, saRNA), and the vaccine is an RNA vaccine. In other aspects, the immunogenic composition comprises DNA, and the vaccine is a DNA vaccine. In other aspects, the immunogenic composition comprises a polypeptide, and the vaccine is a polypeptide vaccine. The 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 infection, cancer, rare diseases, and other diseases or conditions caused by excessive production, insufficient production, or improper production of proteins or nucleic acids.

In some aspects, the composition is substantially free of one or more impurities or contaminants, and, for example, includes nucleic acid or polypeptide molecules that are at least, at most, exactly equal to, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, or any two therebetween; at least 98% pure; or at least 99% pure.

The present disclosure includes methods 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 comprising at least one open reading frame encoding a polypeptide or composition described herein. Thus, the present disclosure encompasses vaccines for active and passive immunization. It is proposed that immunogenic compositions suitable for use as vaccines can be prepared from RNA molecules encoding polypeptides such as VZV glycoproteins. In certain aspects, the immunogenic composition is lyophilized for easier deployment into a desired vehicle.

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 in the form of injectables that are liquid solutions or suspensions; solid forms suitable for forming solutions in liquids or suspensions in liquids prior to injection may also be prepared. The formulation may also be emulsified. The active immunogenic ingredients are typically mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if necessary, the vaccine may contain a large amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, or adjuvants that enhance the effectiveness of the vaccine. In certain aspects, the vaccine is formulated with a combination of substances as described in US Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.

Vaccines can be routinely administered parenterally, for example, by subcutaneous or intramuscular injection. Additional formulations suitable for other modes of administration include suppositories and oral formulations in some cases. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides: such suppositories may be formed from a mixture containing an active ingredient in the range of about 0.5% to about 10%. In some aspects, suppositories may be formed from a mixture containing an active ingredient in the range of about 1% to about 2%. Oral formulations include commonly used excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredients.

Nucleic acid constructs encoding polypeptides and polypeptides can be formulated into vaccines in neutral or salt form. 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 acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid and the like).

Typically, the vaccine is administered in a manner compatible with the dosage form and in an amount such as will be therapeutically effective and immunogenic. The amount to be administered depends on the subject to be treated, including the ability of the subject's immune system to synthesize antibodies and the desired level of protection. The exact amount of active ingredient to be administered is at the discretion of the practitioner. However, a suitable dosage range is about several hundred micrograms of active ingredient per vaccination. The regimen suitable for initial administration and booster injections is also variable, but typically an initial administration is performed, followed by subsequent vaccinations or other administrations.

The mode of administration can vary widely. Any of the conventional methods for administering vaccines can be used. It is believed that these methods include oral administration in a physiologically acceptable solid matrix or parenteral administration by injection or its similar means in a physiologically acceptable dispersion. The vaccine dosage will depend on the route of administration and will vary according to the size and health of the subject.

In some aspects, it will be necessary to administer the vaccine once. In some aspects, it will be necessary to administer the vaccine multiple times, for example, 2, 3, 4, 5, 6 or more times. Vaccinations may be spaced 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11, 12 weeks, including all ranges therebetween. In some aspects, vaccinations may be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, including all ranges therebetween. It may be necessary to periodically boost immunizations at intervals of 1 to 5 years to maintain the protective level of antibodies.

i. Carrier

Pharmaceutically acceptable carriers may include liquid or non-liquid matrices of the composition. If the composition is provided in liquid form, the carrier may be water, such as pyrogen-free water; isotonic saline or buffered (aqueous) solutions, such as phosphate, citrate buffer solutions. Water or buffers containing sodium, calcium and/or potassium salts, such as aqueous buffers, may be used. Sodium, calcium and/or potassium salts may exist in the form of their halides (e.g., chlorides, iodides or bromides), in the form of 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} , _{CaI2 , CaBr2} , _CaCO3 , _CaSO4 , Ca(OH) ₂ . Examples of other carriers may 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; animal fat; 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 oil from cocoa; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid. Examples of other carriers may include colloidal silicon oxide, magnesium stearate, cellulose and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents and the pharmaceutical requirements for their use are described in Remington's Pharmaceutical Sciences.

ii. Adjuvant

Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions. A variety of adjuvants can be used to enhance the antibody response. Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides and natural substances. Specific adjuvants that can be used include Freund's adjuvant, oils such as 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 (such as Pam3Cys). RIBI, which contains three components extracted from bacteria in a 2% squalene/Tween 80 emulsion, namely MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS). Even MHC antigens can be used.

Various methods of achieving an adjuvant effect for vaccines include, respectively, the use of agents such as aluminum hydroxide or aluminum phosphate (alum), which are often used in the form of about 0.05% to about 0.1% solutions in phosphate-buffered saline; and synthetic sugar polymers ( ); aggregation of the proteins in the vaccine by heat treatment at a temperature between about 70°C and about 101°C for a period of 30 seconds to 2 minutes. The following steps can also be used to produce an adjuvant effect: aggregation occurs by reactivation with pepsin-treated (Fab) antibodies against albumin; mixing with bacterial cells (e.g., Cryptosporidium parvum), endotoxins, or lipopolysaccharide components of Gram-negative bacteria; emulsification in a physiologically acceptable oil vehicle (such as mannide monooleate (Aracel A)); or use of perfluorocarbons ( ) was emulsified with a 20% solution of .

In addition to adjuvants, co-administration of biological response modifiers (BRMs) may be necessary 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 cytokines such as gamma-interferon, IL-2, or IL-12, or genes encoding proteins involved in immune helper functions, 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, can also be used in combination with the administration of conventional 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 included are the administration of one or more therapies for treating one or more symptoms of VZV infection, including, but not limited to, steroids (including corticosteroids), anti-inflammatory agents (including acetaminophen or ibuprofen), analgesics, creams or lotions to relieve itching, cold compresses, or various combinations thereof.

In one aspect, it is considered that vaccines and/or therapies are used in combination with antiviral therapy. Alternatively, the therapy may precede or follow another agent treatment at a time interval ranging from a few minutes to a few weeks. In terms of the separate administration of other agents and/or vaccines, it will generally be ensured that the meaningful time period between each delivery is not expired, so that the agent and the immunogenic composition will still be able to exert a favorable combination effect on the subject. In such aspects, it is considered that two modalities may be administered within about 12 to 24 hours of each other or within about 6 to 12 hours of each other. In some cases, it may be necessary to significantly extend the administration period when 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, such as antiviral therapy "A" and immunogenic polypeptides administered as part of an immunotherapy regimen "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 the general regimen for administering such compounds, taking into account the toxicity (if any) of the VZV RNA vaccine composition or other compositions described herein. It is expected that treatment cycles will be repeated as needed. It is also contemplated that different standard therapies (such as moisturizing) may be applied in combination with the described therapies.

D. Application

The administration of the compositions described herein can be carried out via any acceptable mode of administration for medicaments that serve similar functions. In some aspects, the pharmaceutical compositions described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally or intramuscularly. In specific aspects, the VZV RNA molecules and/or RNA-LNP compositions are administered intramuscularly. In certain aspects, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration may include enteral administration (which involves absorption via the gastrointestinal tract) or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than via the gastrointestinal tract, such as by intravenous injection. In one aspect, the pharmaceutical composition is formulated for intramuscular administration. In another aspect, the pharmaceutical composition is formulated for systemic administration, for example, for intravenous administration.

Pharmaceutical compositions can be formulated into preparations in solid, semisolid, liquid, lyophilized, frozen or gaseous forms, 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 so as to allow the active ingredients contained therein to be bioavailable when the composition is administered to the patient. The composition to be administered to the subject or patient is in the form of one or more dosage units, wherein, for example, a tablet can be a single dosage unit, and a container of the compound in the form of an aerosol can accommodate multiple dosage units. The composition to be administered will in any case contain a therapeutically and/or prophylactically effective amount of a compound or a pharmaceutically acceptable salt thereof within the scope of the present disclosure to treat the disease or condition of interest according to the teachings described 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 aspect, the carrier is a microparticle so that the composition is, for example, in the form of a tablet or powder. The carrier may be a liquid so that the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable for administration, for example, by inhalation. In some aspects, when intended for oral administration, the pharmaceutical composition is in solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms included in the forms considered as solid or liquid herein. As a solid composition for oral administration, the pharmaceutical composition may be formulated into powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets, or the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or excluded: a binder such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth gum, or gelatin; an excipient such as starch, lactose, or dextrin; a disintegrant such as alginic acid, sodium alginate, Corn starch and its analogs; lubricants such as magnesium stearate or Slip agents, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), in addition to the above types of substances, it may contain a liquid carrier such as polyethylene glycol or oil. The pharmaceutical composition may be in liquid form, such as an elixir, syrup, solution, emulsion, or suspension. To give two examples, the liquid can be used for oral administration or for delivery by injection. In some aspects, when intended for oral administration, in addition to the compounds of the present disclosure, the composition also contains one or more of a sweetener, a preservative, a dye/colorant, and a flavoring agent. In the composition intended to be administered by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer, and an isotonic agent may or may not be included.

Liquid pharmaceutical compositions, whether in the form of solutions, suspensions or other similar forms, may or may not include one or more of the following adjuvants: sterile diluents such as water for injection, saline solutions (e.g., physiological saline), Ringer's solution, isotonic sodium chloride, fixed oils (such as synthetic monoglycerides or diglycerides that can serve as solvents or suspension media), polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity modifiers such as sodium chloride or dextrose; agents that serve as cryoprotectants such as sucrose or trehalose. Parenteral preparations may be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic. In one aspect, physiological saline is an adjuvant. In one aspect, injectable pharmaceutical compositions are sterile. Liquid pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of the compound such that a suitable dosage will be obtained.

Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical art. For example, a pharmaceutical composition intended for administration by injection can be prepared by combining a nucleic acid or polypeptide with sterile distilled water or other carrier to form a solution. A surfactant can be added to facilitate the formation of a uniform solution or suspension. A surfactant is a compound that non-covalently interacts with a compound consistent with the teachings herein to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.

The pharmaceutical compositions or pharmaceutically acceptable salts thereof according to the present disclosure are generally administered in a "therapeutically effective amount" or a "prophylactically effective amount" and in a "pharmaceutically acceptable formulation". The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredient of the pharmaceutical composition. The terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that achieves a desired response or desired effect alone or together with other doses. In the case of treating a specific disease, in one aspect, the desired response involves inhibiting the course of the disease. This includes slowing the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response for treating a disease may also be delaying the onset of the disease or the condition or preventing the onset of the disease or the condition.

The compositions within the scope of the present disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary depending on various factors, including: the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and duration of action of the therapeutic and/or prophylactic agent; subject parameters of the patient, including the patient's age, weight, general health, sex, and diet; the mode, time, and/or duration of administration; the rate of secretion; the drug combination; the severity of the particular disorder or condition; and the therapy the subject is undergoing. Therefore, the dosage of the compositions described herein may depend on a variety of such parameters. In cases where the response in the patient is inadequate at the initial dose, a higher dose (or an effectively higher dose achieved by another more localized route of administration) may be used. In some aspects, a composition (e.g., VZV RNA-LNP compositions) can be administered at dosage levels sufficient to deliver 0.0001 ng/μg/mg to 100 ng/μg/mg, 0.001 ng/μg/mg to 0.05 ng/μg/mg, 0.005 ng/μg/mg to 0.05 ng/μg/mg, 0.001 ng/μg/mg to 0.005 ng/μg/mg, 0.05 ng/μg/mg to 0.5 ng/μg/mg, 0.01 ng/μg/mg to 50 ng/μg/mg, 0.1 ng/μg/mg to 0.05 ng/μg/mg, 0.05 ng/μg/mg to 0.5 ng/μg/mg, 0.01 ng/μg/mg to 50 ng/μg/mg, 0.1 ng /μg/mg to 40ng/μg/mg, 0.5ng/μg/mg to 30ng/μg/mg, 0.01ng/μg/mg to 10ng/μg/mg, 0.1ng/μg/mg to 10ng/μg/mg, or 1ng/μg/mg to 25ng/μg/mg, once or more a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, preventive, or imaging effect (see, e.g., the unit dose ranges described in International Publication No. WO2013/078199, which is incorporated herein by reference in its entirety). In some aspects, the composition (e.g., VZV RNA-LNP compositions) can be administered at a dosage level sufficient to deliver at least, at most, exactly, or any amount between: 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, 0.00 5. 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, 18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,5 8, 59, 60, 61, 62, 63, 64, 65, 66, 6 7, 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, once or more a day, weekly, monthly, etc., to obtain the desired therapeutic, diagnostic, prophylactic or imaging effect.

In some aspects, a composition (e.g., a VZV RNA-LNP composition) can be administered at a dose level sufficient to deliver at least, at most, just below, or any total dose between: 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 ... 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, 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, 6 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, once or more a day, weekly, monthly, etc., to obtain the desired therapeutic, diagnostic, prophylactic or imaging effect.

In certain aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dose level sufficient to deliver at least, at most, just below, or any total dose between 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.010 001, 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 mg/mL of VZV RNA encapsulated in LNPs.

In exemplary aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dosage level of at least, at most, just below, or any between: 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL of VZV RNA encapsulated in LNP. In exemplary aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dosage level of at least, at most, just below, or any between: 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg of VZV RNA encapsulated in LNP.

In certain aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dose level sufficient to deliver at least, at most, just below, or any total dose between 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.010 001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 μg/mL of VZV RNA encapsulated in LNPs.

In exemplary aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dosage level of at least, at most, just below, or any between: 1, 15, 30, 45, 60, 75, 90, 100, or more micrograms/ml of VZV RNA encapsulated in LNP. In exemplary aspects, the composition (e.g., VZV RNA-LNP composition) can be administered at a dosage level of at least, at most, just below, or any between: 1, 15, 30, 45, 60, 75, 90, 100, or more micrograms of VZV RNA encapsulated in LNP.

The desired dose can be delivered multiple times a day (e.g., 1, 2, 3, 4, 5 or more times a day), every other day, every three days, every week, every two weeks, every three weeks, every four weeks, every 2 months, every three months, every 6 months, etc. In some aspects, the desired dose can be delivered using a single dose administration. In some aspects, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations). When multiple administrations are used, a split dosing regimen can be used. The time between the first administration of a composition and the subsequent administration of the composition may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 37 hours, 38 hours, 39 hours, 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, hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years.

In some aspects, the composition (e.g., VZV RNA-LNP composition) can be administered in a single dose. In some aspects, the composition (e.g., VZV RNA-LNP composition) can be administered twice (e.g., 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), wherein each administration is at least, at most, just below the total dose, or a total dose between any two thereof, or a dose level sufficient to deliver at least, at most, just below the total dose, or a total dose between any two thereof: 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, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 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 of VZV RNA encapsulated in LNP. The present disclosure contemplates higher and lower dosages and frequencies of administration. For example, a composition (e.g., a VZV RNA-LNP composition) can be administered three or four times. Periodic booster immunizations at intervals of 1 to 5 years may be required to maintain protective levels of antibodies.

In some aspects, a composition (e.g., a VZV RNA-LNP composition) is administered to a subject in a single dose of at least, at most, just below, or between any two of: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55 ,56,57,58,59,60,6 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 of VZV RNA encapsulated in LNP. In some aspects, the composition (e.g., VZV RNA-LNP composition) is administered to a subject in a single dose of at least, at most, just below, or between any two of the following: 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of the VZV RNA encapsulated in LNP.

In some aspects, a composition (e.g., a VZV RNA-LNP composition) is administered to a subject at a dose of at least, at most, just below, or between any two of 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 1 5,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55 ,56,57,58,59,60,6 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 of VZV RNA encapsulated in LNP. In some aspects, the composition (e.g., VZV RNA-LNP composition) is administered to a subject at least, at most, just below, or two doses between any two of the following: 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of the VZV RNA encapsulated in LNP.

In particular aspects, a composition (e.g., a VZV RNA-LNP composition) can be administered twice (e.g., day 0 and day 28, day 0 and day 60, day 0 and day 180, day 0 and 2 months later, day 0 and 6 months later), wherein each administration is at least, at most, just below the total dose, or a total dose therebetween, or a dose level sufficient to deliver at least, at most, just below the total dose, or a total dose therebetween: 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more doses of VZV RNA encapsulated in LNPs.

IX. How to use

Provided herein are compositions (eg, pharmaceutical compositions comprising VZV RNA molecules and/or VZV RNA-LNPs), methods, kits, and reagents for preventing and/or treating VZV in humans and other mammals.

VZV RNA compositions (e.g., VZV RNA-LNP compositions) can be used as therapeutic or prophylactic agents. They can be used in medicines to prevent and/or treat infectious diseases. In exemplary aspects, VZV RNA compositions are used to provide prophylactic protection against varicella or herpes zoster. Varicella is an acute infectious disease caused by VZV. The VZV vaccines of the present disclosure can be used to prevent and/or treat VZV (shingles or herpes zoster), and may be particularly suitable for preventing and/or treating immunocompromised and elderly patients to prevent herpes zoster or reduce its severity and/or reduce its duration.

In some aspects, a VZV RNA composition (e.g., a VZV RNA-LNP composition) of the disclosure is administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide.

In some aspects, the VZV RNA compositions of the disclosure can be used to prime immune effector cells, such as activated peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (re-infused) into a subject.

In some aspects, after administering the VZV RNA molecules described herein, for example, formulated as RNA-LNP, at least a portion of the RNA is delivered to the target cell. In some aspects, at least a portion of the RNA is delivered to the cytosol of the target cell. In some aspects, the RNA is translated by the target cell to produce a polypeptide or protein encoded by it. In some aspects, the target cell is a spleen cell. In some aspects, the target cell is an antigen presenting cell in the spleen, such as a professional antigen presenting cell. In some aspects, the target cell is a dendritic cell or a macrophage. RNA molecules (such as RNA-LNP described herein) can be used to deliver RNA to such target cells. Therefore, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, comprising administering RNA particles described herein to the subject.

In some aspects, RNA is delivered to the cytosol of target cells. In some aspects, RNA is translated by target cells to produce RNA-encoded polypeptides or proteins. "Encoding" refers to the intrinsic properties of the templates of other polymers and macromolecules synthesized in biological processes as the specific sequences of nucleotides in the polynucleotides such as genes, cDNA or mRNA, and the polymers and macromolecules have determined nucleotide sequences (such as rRNA, tRNA and mRNA) or determined amino acid sequences and biological properties obtained therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the gene produce proteins in cells or other biological systems, the gene encodes the protein. The nucleotide sequence is consistent with the mRNA sequence and is usually provided in the coding strand in the sequence table and the non-coding strand used as the template for transcription of the gene or cDNA can be referred to as the protein or other products encoding the gene or cDNA.

In some aspects, the nucleic acid compositions described herein, such as 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 aspects, such compositions are characterized in that when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from the human, and in some aspects, such expression persists for a period of at least 36 hours or longer, including, for example, 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 disclosure relates to a method of inducing an immune response against VZV in a subject. The method comprises administering to the subject an effective amount of an RNA molecule, RNA-LNP, and/or composition as described herein.

In another aspect, the disclosure relates to a method of vaccinating a subject. The method comprises administering an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein to a subject in need thereof.

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

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

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

In another aspect, the disclosure relates to a method of treating or preventing VZV infection and/or a disease caused by VZV or reducing the severity of a subject by, for example, inducing an immune response to VZV in the subject. In some aspects, the method comprises administering a primary immunization composition comprising an effective amount of RNA molecules, RNA-LNPs, and/or compositions as described herein, and administering a booster composition comprising an effective amount of RNA molecules, RNA-LNPs, and/or compositions as described herein. In some aspects, the composition elicits an immune response comprising an antibody response. In some aspects, the composition elicits an immune response comprising 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 specific for a VZV antigenic polypeptide in the subject, wherein the anti-antigenic polypeptide antibody titer in the subject after vaccination is increased relative to the anti-antigenic polypeptide antibody titer in a subject vaccinated with a prophylactically effective dose (e.g., a therapeutically effective dose that prevents viral infection at a clinically acceptable level) of a traditional vaccine for VZV. "Anti-antigenic polypeptide antibodies" are serum antibodies that specifically bind to an antigenic polypeptide. In some aspects, after administration of the VZV RNA-LNP composition, the anti-antigenic polypeptide antibody titer in the subject is increased by at least the following number, at most the following number, any two of the following number, or exactly the following number: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 logs relative to the anti-antigenic polypeptide antibody titer in a subject administered a prophylactically effective dose of a traditional composition for VZV.

The methods disclosed herein may involve administering to a subject a VZV RNA-LNP composition comprising at least one VZV having an open reading frame encoding at least one VZV antigenic polypeptide. RNA molecules, thereby inducing an immune response specific for a VZV antigenic polypeptide in a subject, wherein the immune response in the subject is equivalent to the immune response in a subject administered with at least the following amounts, at most the following amounts, any amounts therebetween, or exactly the following amounts of a traditional composition for VZV: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100 times the dose level of the RNA composition.

In some aspects, RNA molecules, RNA-LNPs and/or compositions are used as vaccines. In some aspects, RNA molecules, RNA-LNPs and/or compositions can be used in various treatment or prevention methods for preventing, treating or ameliorating herpes zoster or shingles or conditions associated with herpes zoster or shingles.

In some aspects, the RNA molecules, RNA-LNPs and/or compositions can be used in various therapeutic or preventive methods for preventing, treating or ameliorating postherpetic neuralgia.

VZV RNA compositions can be administered prophylactically or therapeutically to healthy subjects or early in infection, during the incubation period, or during active infection after symptom onset. In some aspects, the subject is immunocompetent. In some aspects, the subject is immunocompromised.

In some aspects, the RNA molecules, RNA-LNPs and/or compositions are administered in a single dose. In some aspects, a second, third or fourth dose may be given. In some aspects, the RNA molecules, RNA-LNPs and/or compositions are administered in multiple doses.

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

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

In some aspects, the RNA molecules, RNA-LNPs and/or compositions described herein are administered to a subject less than about 1 year old, or about 1 year old to about 10 years old, or about 10 years old to about 20 years old, or about 20 years old to about 50 years old, or about 60 years old to about 70 years old, or older.

In some aspects, the age of the subject is at least, at most, just below, or between any two of less than 1 year old, greater than 1 year old, greater than 5 years old, greater than 10 years old, greater than 20 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, or older. In some aspects, the subject is greater than 50 years old.

In some aspects, the age of the subject is at least, at most, just below, 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 aspects, the subject may be about 50 years or older.

In some aspects, the age of the subject is at least, at most, just below, or between any two of: 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 older. In some aspects, the subject may be 50 years or older.

X. Clinical Research

The VZV RNA-LNP vaccine of the present disclosure comprises a nucleoside-modified mRNA (modified RNA; modRNA) encoding glycoprotein E (gE) from VZV. The VZV RNA-LNP vaccine may comprise RNA containing a single-stranded, 5'-capped and polyadenylated modified RNA, which is translated after entering the cell. The RNA comprises an open reading frame (ORF) encoding a variant of VZV gE. For example, the RNA molecule may comprise gE_WT CO2 (RNA encodes a full-length gE protein located in the plasma membrane and the Golgi apparatus), gE ms5 CO1 (RNA encodes a C-terminally truncated gE protein mainly located in the plasma membrane) and/or gE ms6 CO2 (RNA encodes the extracellular domain of a secreted gE protein). In addition, as described herein, the RNA may comprise structural components optimized for high potency of the RNA, such as untranslated regions (UTRs). The VZV RNA-LNP may comprise RNA as provided in Table 5 of Example 1 disclosed herein. VZV RNA-LNPs may comprise RNA as provided in Tables 1 to 3 of Example 7 disclosed herein. The RNA may also comprise substitution of uridine with 1-methyl-pseudouridine to reduce recognition of the vaccine RNA by innate immune sensors such as toll-like receptors (TLRs) 7 and 8, thereby resulting in a decrease in innate immune activation and an increase in protein translation.

The RNA molecules described herein are formulated/encapsulated in lipid nanoparticles (LNPs) to enable delivery of RNA to host cells after intramuscular (IM) injection. LNP formulations 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 RNA from extracellular RNase degradation and facilitates delivery in cells. After IM injection of VZV RNA-LNP vaccines, LNPs are absorbed by cells and RNA is released into the cytosol. In the cytosol, RNA is translated and the encoded viral antigens are produced.

The examples herein demonstrate that the VZV RNA-LNP vaccines of the present disclosure are immunogenic in mice and induce humoral and cell-mediated immune responses in mice.

The clinical studies of the present disclosure evaluate the safety, tolerability and immunogenicity of VZV RNA-LNP vaccines against VZV. For example, VZV RNA-LNP vaccines may be suitable for active immunization for the prevention of herpes zoster disease caused by VZV in adults (e.g., age ≥45, ≥50, ≥55, ≥60, ≥70, etc., or 50 to 69 years old). VZV RNA-LNP vaccines may be administered at different dose levels, dosage forms, dosing times and dosing schedules as described herein, including (but not limited to):

- Single dose schedule or 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-LNP can be presented in liquid or lyophilized formulations. Administration of the VZV RNA-LNP vaccine can be administered in the following range: about 15 μg, about 30 μg, about 60 μg, about 90 μg, about 100 μg or higher doses per administration with an injection volume of about 0.25 to 1 mL (e.g., about 0.25, 0.5, 1 mL). Dilution with sterile 0.9% sodium chloride (standard saline) may be required.

Objectives of VZV RNA-LNP clinical studies may include (but are not limited to):

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

-Describe the dose level and schedule of and the immune responses elicited by the VZV RNA-LNP vaccine in participants.

Example

Below is an embodiment for implementing the specific aspects of the present disclosure. The following examples are included to illustrate 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 will be appreciated by those skilled in the art that the technology disclosed in the following examples represents the technology that the inventors have found to work well in the practice of the present disclosure. However, it will be appreciated by those skilled in the art that, according to the present disclosure, many changes can be made to the disclosed specific aspects without departing from the spirit and scope of the present disclosure and still obtaining the same or similar results. Efforts have been made to ensure the accuracy of the numbers used (e.g., amount, temperature, etc.), but some experimental errors and deviations should certainly be allowed.

Example 1. Generation of VZV RNA Constructs

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

Table 4. VZV gE proteins and descriptions

DNA sequences encoding VZV proteins were prepared and used in an in vitro transcription reaction to produce RNA. In vitro transcription of RNA is known in the art and described herein. The DNA template was cloned into a plasmid vector with backbone sequence components (T7 promoter, 5' and 3' UTRs, polyadenylic acid tail to improve RNA stability and translation efficiency). The DNA was purified, quantified spectrophotometrically, and in vitro transcribed by T7 RNA polymerase in the presence of a trinucleotide end cap 1 analog (( _m27,3' ^-O )Gppp( ^{m2' -O} )ApG) (TriLink) and in which N1-methyl pseudouridine (Ψ) replaced uridine (modified RNA; modRNA).

VZV RNA is produced from codon optimized (CO) DNA to achieve stability and excellent protein expression. As used herein, CO1 indicates about 58% G/C content, CO2 indicates about 66% G/C content, and CO3 indicates about 62% G/C content. Table 5 shows RNA constructs and corresponding sequences of the present disclosure, including 5'UTR, an open reading frame encoding a varicella zoster virus (VZV) polypeptide, 3'UTR, and a polyadenylation tail.

Table 5. VZV gE RNA constructs/molecules

*The poly(A) tail length can contain +2/-2A or +1/-1A.

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

Example 2. VZV gE expression and subcellular localization (Vero cells)

Expression of VZV RNA constructs was tested in transfected Vero cells (a renal epithelial cell culture line derived from African green monkeys). Inoculated Vero cells were transfected with 10 ng, 25 ng, or 50 ng of RNA constructs (Table 5) at 37°C, 5% _CO2 for 24 hours using MESSENGERMAX ^™ according to the manufacturer's instructions. Cells were washed three times with PBS+Ca/Mg and fixed in 4% paraformaldehyde (PFA) at 25°C for 20 minutes. Cells were washed twice with PBS+Ca/Mg containing 3% bovine serum albumin (BSA). Primary and secondary staining antibodies were diluted in goat serum containing 0.1% saponin. Cells were stained with primary antibodies diluted 1:1000 for 1 hour at 37°C, washed three times with PBS+Ca/Mg, and incubated with secondary antibodies (1:500) and CELLMASK ^™ (1:140,000) at 37°C for 45 minutes. The cells were then stained with Dapi (1:15,000 to 1:20,000) for 15 min at 25°C and washed three times with PBS+Ca/Mg. Plus High Content Screening System analyzes cells for VZV gE expression and subcellular localization at 10x or 63x magnification.

Imaging analysis showed a dose-dependent increase in the percentage of Vero cells transfected with VZV gE ⁺ , with almost 100% of cells expressing VZV gE at a dose of 50 ng in the cytoplasmic tail mutant (Fig. 2) and about 80% of cells expressing VZV gE at a dose of 50 ng in the secretory mutant (Fig. 3). Since the secretory mutant is primarily transported to the outside of the cell, lower levels were detected inside.

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

Imaging analysis shows the subcellular localization of VZV gE in Vero cells transfected with various RNA constructs. The localization of VZV gE in cells transfected with gE_WT appears in the cell membrane and the mature network of the Golgi apparatus (TGN; Figure 6, 63 times magnification; Figure 10, 10 times magnification). The cytoplasmic tail mutants (ms4, ms5, ms8, ms9, ms10, ms11, ms12) preferentially show VZV gE localization in the cell membrane (Figures 6 to 8, 63 times magnification; Figures 10 to 12, 10 times magnification). The secretory mutants (ms3, ms6) show VZV gE localization in the culture supernatant (Figure 9, 63 times magnification; Figure 13, 10 times magnification).

Example 3. Preparation of VZV gE modRNA formulated in LNP

Purified RNA (as described in Table 5) was formulated/encapsulated in lipid nanoparticles (RNA-LNPs) using an ethanol lipid mixture of ionizable cationic lipids and transferred to an aqueous buffer system via diafiltration to obtain a lipid nanoparticle composition as described herein. The RNA-LNPs comprised VZV RNA molecules, cationic lipids, ((4-hydroxybutyl) azadialkyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), PEGylated lipids, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl) acetamide, and two structural lipids (1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol), see Table 6.

Table 6. Lipid formulations

Example 4: In vitro expression (HEK 293T cells)

In vitro expression (IVE) analysis was performed to assess the potency of the VZV RNA-LNP vaccine. RNA constructs were formulated into 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 at 37°C, 5% _CO2 for 24 hours. The transfected cells were washed with DPBS and incubated with Strip from the plate wells and rinse with cold PBS. Cells were subjected to live/dead staining, fixation and permeabilization before FACS staining. Fixed and permeabilized cells were incubated with anti-gE primary antibody (1:1000) at 2 to 8 ° C for 45 minutes, washed twice with BD PERM/WASH ^TM buffer, and incubated with goat anti-human κ-PE secondary antibody (1:1000) at 2 to 8 ° C for 30 minutes. Cells were washed twice with BD PERM/WASH ^TM buffer, resuspended in 1×FACS buffer, and analyzed on BD LSRII to determine the cell ratio of VZV gE expressed for each VZV RNA-LNP input. A titration curve was created, in which the input RNA amount was drawn for the percentage of cells expressing gE 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 the MFI of VZV RNA-LNP (293T cells) and VZV gE RNA (Vero cells; see Example 2) showed that VZV RNA-LNP and VZV gE RNA shared similar MFI trends (Table 7), with gE_WT CO2>gE_WT CO1>ms4 CO1>ms3 CO1 for potency and MFI.

Table 7. In vitro expression

* Secretory

This trend is also observed in the data of Table 8 and Figure 15, where for efficacy and MFI, ms5CO1>gE_WT CO2>gE_WT CO1>ms6 CO2.

Table 8. In vitro expression

* Secretory

Example 5. Immune response (in vivo experiment)

The VZV RNA-LNP vaccine was tested for its ability to induce IgG and T cell responses in Balb/c mice. Fifteen 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. Immunized mice 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 group on days 0 and 28. Serum was collected from mice on days 28 and 42 for characterization of IgG levels binding to VZV gE ( Spleens were collected from five mice in each group on day 35 and serum was collected for cellular analysis.

Table 9. and the administration schedule of VZV RNA-LNP

*5 mice were euthanized on day 34 to obtain spleens (T cells)

IgG antibody titer

use The serum IgG level of each mouse sample was measured by the platform. Briefly, the diluted serum was incubated at 2 to 8°C and 300 rpm on a blocking recombinant gE protein-bound monolayer microsphere ( Microspheres, region 79) in the dark for 20 ± 4 hours. Secondary antibody solution (R-phycoerythrin conjugated affinity purified F(ab)'2 fragment goat anti-mouse IgG F(ab)'2 fragment specific) was prepared at 1:250 in LXA-16 buffer. The well plate was washed three times in LXA-20 (EIA-7) and the secondary antibody was applied to each well of the assay plate. The well plate was covered with an aluminum seal and incubated at 25°C, 300 rpm for 2 hours ± 15 minutes. The well plate was washed three times with LXA-20 (EIA-7) buffer and 100 μl of LXA-20 was applied to each well. The well plate was covered with an aluminum seal and incubated at 25°C, 300 rpm for a minimum of 4 minutes or a maximum of 2 hours. In BIO- The plates were analyzed on a reader. IgG titers on days 28, 34 and 42 are shown in Table 10 and Figures 17-19.

Table 10. Geometric mean titers (GMC) on days 28, 34 and 42

As shown in Figure 16, IgG titers on day 28 (prime vaccination only/before booster vaccination) showed that Dose-dependent changes in the geometric mean concentration (GMC) of gE_WT CO1 and lyophilized gE_WT CO1 in mice. The GMC of gE_WT CO1 (7.69) and the GMC of gE_WT CO2 (12.90) administered at 0.5 μg (1/60 of the potential clinical dose of 30 μg) were significantly higher than those administered at 1 μg (1/50 of its human clinical dose of 50 μg). The GMC (2.33) of gE_WT CO1 administered at 1 μg (1/60 of the potential clinical dose of 60 μg) was higher than that of gE_WT CO1 administered at 1 μg (1/50 of its human clinical dose of 50 μg). GMC(2.33).

As shown in Figure 17, the IgG titers on day 34 (six days after the booster immunization on day 28) also showed that The dose-dependent changes of GMC in mice treated with gE_WT CO1 and freeze-dried gE_WT CO1 (Figure 17). In contrast, higher IgG levels were observed for the VZV RNA-LNP vaccines (specifically, GMC 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, relative to GMC of 437.9 for gE_WT CO2 at 1/50 of its human clinical dose). The GMC is 177.9 (Figure 17).

When comparing gE_WT CO1 (about 58% G/C) and gE_WT CO2 (about 66% G/C), a positive correlation between higher G/C content and higher IgG titer was observed, see Figures 16 and 17.

As shown in Figure 18, the IgG titer on day 42 further showed a dose-dependent change in geometric mean concentration (GMC). (GMC 498.79), higher IgG levels were observed in gE_WT CO1 (GMC 918.43) and gE_WT CO2 (GMC 524.39).

Figure 19 shows the observed levels of leukemia at day 28 (primary vaccination only), day 34 (six days after booster vaccination), and day 42. Summary of IgG levels of the 1 μg and RNA-LNP vaccines (1 μg).

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

Cell-mediated immunity (T cell response)

At the 34th day (34 days after immunization, 6 days after booster immunization), spleen cells were collected from Balb/c mice to assess the induced gE-specific T cell response. Intracellular cytokine staining (ICS) analysis was used to detect the presence of cytokines in CD4 ⁺ or CD8 ⁺ T cells after antigenic peptide stimulation. ICS analysis can detect the various cytokines produced in CD4 ⁺ and CD8 ⁺ T cells after antigenic peptide stimulation, including IFN-γ. During in vitro stimulation of spleen cells, reagents for blocking protein secretion were added to retain the synthesized cytokines, thereby allowing them to be detected by intracellular staining. After stimulation, cells were stained for surface and intracellular markers to identify T cell types (CD3 ⁺ cells of CD4 and CD8 T cells), activation markers (CD154/CD40L) and cytokines. CD4 ⁺ T cells expressing IFN-γ, IL-2, TNFα and CD40L and CD8+ T cells expressing IFN-γ were assessed to assess gE-specific T cells.

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

As shown in FIG. 20 , examination of CD4 ⁺ IFN-γ ⁺ (Th1) T cells showed a strong dose-dependent response in the groups receiving VZV RNA-LNP vaccines (including lyophilized vaccines) and As shown in Figure 21, examination of CD8 ⁺ IFN-γ ⁺ T cells showed strong but variable responses in the group receiving the VZV RNA-LNP vaccine and No detectable response in mice.

Example 6. Immune Response - Mice Experienced with LAV (In Vivo)

The ability of VZV RNA-LNP vaccines to induce IgG and T cell responses in C57BL/6 mice was tested. Fifteen mice per group were immunized according to the schedule and specifications in Table 11. Briefly, 10 or 15 mice per group were immunized on day 0 with live attenuated varicella (LAV) vaccine ( Merck) used a full human dose (1350 pfu) per mouse for subcutaneous (SQ) priming, intramuscular (IM) immunization on day 35, and boosting on day 63. The "infection" with the LAV vaccine mimics the VZV exposure that humans receive when they are infected with VZV as children and develop varicella.

use Immunized mice 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 μg VZV RNA-LNP; mice immunized with ms6 CO2 received 0.5 or 1 μg VZV RNA-LNP; and mice immunized with lyophilized gE_WT CO2 received 0.5 or 1 μg VZV RNA-LNP. Saline was administered to the negative control group on days 35 and 63. Serum was collected from mice on days 35, 63 and 76 for characterization of IgG levels binding to VZV gE ( Spleens were collected on days 48 (13 days after the first dose for selected groups) and 76 for cellular analysis (T and B cell responses).

Table 11. and the administration schedule of VZV RNA-LNP

* Spleen collection, day 48: 8 groups, 40 mice; day 76: 10 groups, 50 mice

** (LAV) was administered at full clinical dose (1350 pfu) in a total volume of 0.5 mL on day 0

IgG antibody titer

use The serum IgG level of each mouse sample was determined by the platform (described herein). IgG titers on days 35, 63 (1 month after the first dose) and 76 (13 days after the second dose) are shown in Table 12 and Figures 22 to 24. Each data point in the figure is the result of a single animal; each horizontal line represents the geometric mean IgG concentration (μg/ml) and the whiskers represent the 95% confidence interval.

Table 12. Geometric mean titer (GMC) on day 35, day 63 and day 76

As expected before immunization, Figure 22 shows that on day 0, the LAV vaccine ( ) low IgG titer level on day 35 after SQ priming. As shown in Figure 23, after receiving VZV RNA-LNP vaccine or In LAV-experienced mice, the IgG titers increased significantly on day 63 (1 month after the first dose) in a dose-dependent response. FIG. 24 shows that the IgG titers increased further on day 76 (13 days after the second dose/boost immunization) in a dose-dependent response.

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

Cell-mediated immunity (T cell and B cell responses)

After stimulating splenocytes in vitro with a pool of gE peptides (each peptide is 2 μg/mL), vaccine-induced T cell responses were measured by ELISpot and intracellular cytokine staining (ICS) analysis (described herein). For ELISpot, the cytokine IFN-γ secreted by activated T cells was captured by anti-IFN-γ antibodies on a polyvinylidene fluoride (PVDF) membrane coated at the bottom of the well on a microplate. Anti-IFN-γ secondary antibodies were labeled with another non-competitive biotin, followed by an enzyme color reaction using a streptavidin alkaline phosphatase (ALP) conjugate and substrate solution, nitro blue tetrazolium, and 5-bromo-4-chloro-3'-indolyl phosphate (BCIP/NBT-plus) that produced a dark purple precipitate or spot, so that the captured IFN-γ produced spots. Mabtech mouse IFN-γELISpot PLUS kit (ALP) was used to measure T cell IFN-γ responses, and it was expressed as spot-forming cells (SFC) per million cells. The ICS assay measures IFN-γ expressing cells within CD4 ⁺ and CD8 ⁺ T cells, which is expressed as the percentage of IFN-γ ⁺ cells within CD4 ⁺ and CD8 ⁺ T cells.

Vaccine-induced B cell responses were assessed by measuring the frequency of VZV gE-specific B cells in the spleen. The wild-type gE protein ectodomain with a streptag was coupled to streptavidin (SA)-fluorescent dyes PE and APC (both from BioLegend) at a 2:1 ratio for 1 hour at room temperature (RT) in a separate tube at 20× the desired staining concentration (10 μg/mL for each protein). Each SA-fluorescent dye-coupled spike protein was pooled and diluted to 1× using flow cytometry (FC) buffer (2% FBS/PBS) to generate a B cell probe for identifying gE-specific B cells. Single cell suspensions of splenocytes (5×10 ⁶ cells/well) were first washed in PBS and stained with eFluor 506 Fixable Viability dye for 10 minutes at room temperature to distinguish live from dead cells. After washing in FC buffer, cells were incubated with 50 μL/well of 1×B cell probe for 30 to 45 minutes and then washed to remove unbound probe. Cells were surface stained with a mixture of flow cytometry antibodies (CD19, IgD, IgM, IgD, all from BioLegend) to identify B cell surface phenotypes. After washing, cells were fixed using BD fixation buffer and suspended in FC buffer. Cells were collected on LSR Fortessa and data were analyzed by FlowJo (10.7.1). The results of gE-specific B cells are expressed as the percentage of B cells expressing IgG.

Day 48

Splenocytes were collected from C57BL/6 mice on day 48 (13 days after the first dose) to assess the gE-specific cellular immune response induced after a single vaccine dose, see Table 13 and Figures 26A to 26D. or VZV RNA-LNP vaccine was immunized IM, and spleens were collected on day 48 (13 days after the first dose).

Table 13. Cellular immune response on day 48 (13 days after the first dose)

Figure 26A shows that after a single vaccine dose, mice subjected to LAV elicited a strong gE-specific IFN-γ (T cell) response in a dose-dependent manner, as measured by IFN-γ ELISpot. As shown in Figure 26B, ICS analysis results showed that the vaccine induced a similar strong dose-dependent gE-specific IFN-γ ⁺ CD4 ⁺ T cell response, as demonstrated by ELISpot. Figure 26C shows that gE-specific IFN-γ ⁺ CD8 ⁺ T cell responses were induced only by VZV RNA-LNP vaccines (gE_WT CO2, ms5CO1, ms6CO2, gE_WT CCO2 lyo), but not by Induction, indicating that the VZV-RNA-LNP vaccine induces a unique immune response. Overall, on day 48 (13 days after the first dose), the T cell response induced by 1 μg (1/60 of the potential clinical dose of 60 μg) of gE_WT CO2 was significantly higher than that induced by 1 μg (1/50 of its human dose) Induced T cell responses.

B cell responses in splenocytes were assessed by measuring the frequency of gE-specific IgG ⁺ B cells by flow cytometry. As shown in Figure 26D, the frequency of gE-specific IgG ⁺ B cells showed that the B cell response induced by the vaccine was similar to the gE-specific IFN-γ ⁺ CD4 ⁺ T cell response.

Day 76

Splenocytes were collected from C57BL/6 mice on day 76 (13 days after the second dose/boost) to assess gE-specific cellular immune responses induced after the second/boost vaccine dose, see Table 14 and Figures 27A to 27C. or VZV RNA-LNP vaccine was immunized IM, and spleens were collected on day 76 (13 days after the second dose/boost).

Table 14. Cellular immune responses on day 76 (13 days after the second dose/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 analysis. As shown in Figure 27B, a steady increase in gE-specific IFN-γ ⁺ CD8 ⁺ T cell responses was induced only by VZV RNA-LNP vaccines (gE_WT CO2, ms5 CO1, ms6 CO2, gE_WT CCO2 lyo), but not by Induction, confirming that the VZV-RNA-LNP vaccine induces a unique immune response. Overall, at day 76 (13 days after the second dose/boost), the T cell response induced by 1 μg (1/60 of the potential clinical dose of 60 μg) of gE_WT CO2 was higher than that induced by 1 μg (1/50 of its human dose) Induced T cell responses.

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

Example 7. VZV antigen

The sequences of VZV antigens/polypeptides, VZV DNA and VZV RNA of the present disclosure are provided in Tables 1 to 3. The sequences may include any stop codon, including but not limited to the stop codons provided in the Tables.

Table 1. VZV polypeptides

Table 2. VZV DNA

Table 3. VZV RNA

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 of paragraphs 1 to 4, wherein the VZV polypeptide is a full-length VZV polypeptide, a truncated VZV polypeptide, a fragment of a VZV polypeptide, or a variant of a VZV polypeptide.

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

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

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

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

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

11. The RNA molecule of any of paragraphs 1 to 10, wherein the open reading frame is transcribed from a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of any of SEQ ID NOs: 12 to 145.

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

13. The RNA molecule of any of paragraphs 1 to 12, wherein the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence of any one of SEQ ID NOs: 146 to 279.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

37. The RNA molecule of any 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 to 313.

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

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

41. The RNA molecule of any of paragraphs 1 to 40, wherein the RNA molecule comprises a 5' terminal cap moiety.

42. The RNA molecule of paragraph 41, wherein the RNA molecule comprises a 5' end cap portion comprising (3'OMe)-m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG.

43. The RNA molecule of any of paragraphs 1 to 42, further comprising a 3' poly(A) tail.

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

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

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

47. The RNA molecule of any of paragraphs 1 to 46, wherein the RNA molecule comprises a 5'UTR and a 3'UTR.

48. The RNA molecule of any of paragraphs 1 to 47, wherein the RNA molecule comprises a 5' end cap, a 5' UTR and a 3' UTR.

49. The RNA molecule of any of paragraphs 1 to 48, wherein the RNA molecule comprises a 5' end cap, a 5' UTR, a 3' UTR and a poly(A) tail.

50. The RNA molecule of any 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 NO: 146 to 279; and a 3'UTR comprising the sequence of SEQ ID NO: 284 or 317.

51. An RNA molecule as described in any of paragraphs 1 to 50, comprising: a 5'UTR comprising the sequence of any one of SEQ ID NOs: 28, 312 and 313; an open reading frame comprising the sequence of any one of SEQ ID NOs: 146 to 279; a 3'UTR comprising the sequence of any one of SEQ ID NOs: 284, 314 and 317; and a poly(A) tail comprising the sequence of any one of SEQ ID NOs: 287 and 315.

52. The RNA molecule of any of paragraphs 1 to 51, wherein the open reading frame is produced by codon-optimized DNA.

53. The RNA molecule of any 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% to 75%, about 55% to 70%, about 58%, about 66%, or about 62%.

54. The RNA molecule of any of paragraphs 1 to 53, wherein the encoded VZV polypeptide is located in the cell membrane, in the Golgi apparatus and/or is anchored in the membrane and secreted.

55. The RNA molecule of any of paragraphs 1 to 54, wherein the RNA molecule comprises stabilized RNA.

56. The RNA molecule of any 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-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine.

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

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

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

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

62. A composition comprising the RNA molecule of any 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 structured lipid.

64. The composition of paragraph 62 or 63, wherein the lipid nanoparticle comprises a cationic lipid.

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

66. The composition of any of paragraphs 61 to 64, wherein the lipid nanoparticle comprises a pegylated lipid.

67. A composition as described in any of paragraphs 63 to 66, wherein the PEGylated lipid is a PEGylated phosphatidylethanolamine, a PEGylated phosphatidic acid, a PEGylated ceramide (e.g., PEG-CerC14 or PEG-CerC20), a PEGylated dialkylamine, a PEGylated diacylglycerol, a PEGylated dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide, a glycol lipid (including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxypropane-3-amine (PEG-c-DMA) and PEG-2000-DMG), a PEGylated diol acylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((o-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(u>-methoxy(polyethoxy)ethyl)carbamate.

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

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

70. The composition of paragraph 69, wherein 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 dioleoylphosphatidylcholine (DOPC). Oleyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-ditransoleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE).

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 of paragraphs 63 to 71, wherein the second structured 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 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 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 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) azadialkyl) 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-di(tetradecyl)acetamide (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 of paragraphs 62 to 76, comprising the RNA molecule at a concentration of about 0.06 mg/mL, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP).

78. The composition of any 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 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 Tris buffer.

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

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

83. The composition of paragraph 81 or 82, wherein the concentration of the Tris HCl is about 1.25 to 1.40 mg/mL or about 0.5 to 0.65 mg/mL.

84. The composition of any of paragraphs 78 to 83, wherein the stabilizer is sucrose.

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

86. The composition of any of paragraphs 78 to 85, wherein the salt diluent used for reconstitution is sodium chloride.

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

88. The composition of any of paragraphs 62 to 87, wherein the composition is liquid or lyophilized.

89. A composition as described in paragraph 62, comprising an RNA molecule at a concentration of about 0.01 to 0.09 mg/mL, the RNA molecule being 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; and the composition further comprises a Tris buffer containing tromethamine at a concentration of about 0.1 to 0.3 mg/mL and Tris hydrochloric acid (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, wherein the composition is a liquid composition.

90. The composition of paragraph 62, comprising an RNA molecule at a concentration of about 0.01 to 0.09 mg/mL, the RNA molecule being 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; and the composition further comprises a Tris buffer containing tromethamine at a concentration of about 0.01 to 0.15 mg/mL and Tris hydrochloric acid (HCl) at a concentration of about 0.5 to 0.65 mg/mL, and sucrose at a concentration of about 35 to 50 mg/mL.

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

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

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

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

95. 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 composition as described in any 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 as described in any of paragraphs 1 to 93 for the preparation of a medicament for inducing an immune response against VZV in a subject.

100. Use of an RNA molecule or composition as described in any of paragraphs 1 to 93 for the preparation of a medicament for preventing, treating or ameliorating 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 of paragraphs 94 to 103, wherein the subject is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older.

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

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

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

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

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

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

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

113. The method or use of any of paragraphs 94 to 110, wherein two doses of the RNA molecule and/or composition are administered to the subject, on day 0 and about 2 months later.

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

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

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

All methods disclosed and advocated herein can be performed and executed without undue experimentation in accordance with the present disclosure. Although the compositions and methods of the present disclosure have been described according to certain aspects, it will be apparent to those skilled in the art that variations may be applied to the methods described herein and the steps of the methods or the order of steps of the methods without departing from the concept, spirit and scope of the present disclosure. More specifically, it is apparent that certain reagents that are chemically and physiologically related may replace the reagents described herein while achieving the same or similar results. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

All cited references recited in this application (including literature references, issued patents, published patent applications, and The contents of the WO 2009/0392, ...10/0392, WO 2011/0392, WO 2012/0392, WO 2013/0392, WO 2014/0392, WO 2015/0392, WO 2016/0392, WO 201

Claims (32)

1. An RNA molecule comprising at least one open reading frame encoding a varicella zoster virus (VZV) glycoprotein E (gE) polypeptide. 2. The RNA molecule of claim 1, wherein the VZV polypeptide is a full-length VZV polypeptide, a truncated VZV polypeptide, a fragment of a VZV polypeptide, or a variant of a VZV polypeptide. 3. The RNA molecule of claim 1 or 2, wherein the VZV polypeptide comprises at least one mutation. 4. The RNA molecule of any one of claims 1 to 3, wherein the VZV polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the amino acid sequences selected from SEQ ID NOs: 1 to 11. 5. The RNA molecule of any one of claims 1 to 4, wherein the open reading frame is transcribed from a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the sequences selected from SEQ ID NOs: 12 to 145. 6. The RNA molecule of any one of claims 1 to 5, wherein the open reading frame comprises a nucleic acid sequence that is at least 90%, 95%, 96%, 97%, 98% or 99% identical to any one of the sequences selected from SEQ ID NOs: 146 to 279. 7. The RNA molecule according to any one of claims 1 to 6, wherein the open reading frame comprises a nucleic acid sequence selected from any one of SEQ ID NOs: 146 to 279. 8. The RNA molecule according to any one of claims 1 to 7, further comprising a 5' untranslated region (5'UTR). 9. The RNA molecule of claim 8, wherein the 5'UTR comprises a sequence selected from any one of SEQ ID NO: 281, 312 or 313. 10. The RNA molecule according to any one of claims 1 to 9, further comprising a 3' untranslated region (3'UTR). 11. The RNA molecule of claim 10, wherein the 3'UTR comprises a sequence selected from any one of SEQ ID NO: 284, 314 or 317. 12. The RNA molecule according to any one of claims 1 to 11, wherein the RNA molecule further comprises a 5' cap moiety and/or a 3' poly(A) tail. 13. An RNA molecule as claimed in claim 12, wherein the poly(A) tail comprises a sequence selected from any one of SEQ ID NO: 287 or 315. 14. The RNA molecule of any one of claims 1 to 13, 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% to 75%, or about 55% to 70%. 15. The RNA molecule according to any one of claims 1 to 14, wherein the encoded VZV polypeptide is localized in the cell membrane, in the Golgi apparatus and/or is secreted. 16. The RNA molecule according to any one of claims 1 to 15, wherein the RNA comprises at least one modified nucleotide. 17. The RNA molecule of any one of claims 1 to 16, wherein each uridine is replaced by N1-methylpseudouridine (Ψ). 18. The RNA molecule according to any one of claims 1 to 17, wherein the RNA is mRNA. 19. A composition comprising the RNA molecule of any one of claims 1 to 18, wherein the RNA molecule is formulated in a lipid nanoparticle (LNP). 20. The composition of claim 19, wherein the lipid nanoparticles comprise at least one of a cationic lipid, a pegylated lipid, a neutral lipid, and a steroid or a steroid analog. 21. The composition of claim 20, wherein the cationic lipid is (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315). 22. The composition of claim 20 or 21, wherein the PEGylated lipid is a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159), a glycol lipid (including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000) carbamoyl]-1,2-dimyristoyloxypropane-3-amine (PEG-c-DMA) and PEG-2000-DMG), a poly [00136] The invention also provides a method for preparing the present invention which can be used to prepare the present invention. The invention can be used to prepare the present invention. The invention can be used to prepare the present invention. 23. The composition of any one of claims 20 to 22, wherein 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 dioleoylphosphatidylcholine (POPC). Acyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), or 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). 24. A composition according to any one of claims 20 to 23, wherein the steroid or steroid analogue is cholesterol. 25. A method of inducing an immune response against VZV in a subject, comprising administering to the subject an effective amount of an RNA molecule or composition according to any one of claims 1 to 24. 26. A method for preventing, treating or ameliorating a VZV-associated infection, disease or condition in a subject, comprising administering to the subject an effective amount of an RNA molecule or composition according to any one of claims 1 to 24. 27. Use of an RNA molecule or composition as claimed in any one of claims 1 to 24 for the preparation of a medicament for preventing, treating or ameliorating a VZV-associated infection, disease or condition in a subject. 28. The method or use of any one of claims 26 or 27, wherein the infection, disease or condition is herpes zoster (shingles) or post-herpetic neuralgia. 29. Use of an RNA molecule or composition as claimed in any one of claims 1 to 24 for the preparation of a medicament for inducing an immune response against VZV in a subject. 30. The method or use of any one of claims 25 to 29, wherein the subject is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. 31. The method or use of any one of claims 25 to 30, wherein the RNA molecule or composition is administered as a vaccine. 32. The method or use of any one of claims 25 to 30, wherein a single dose, two doses, three doses or more and optionally a booster dose of the RNA molecule or composition is administered to the subject.