mRNA COMPOSITIONS AND USES THEREOF IN VARICELLA ZOSTER VIRUS VACCINES

Abstract

The present disclosure relates to mRNA sequences encoding an antigen from varicella zoster virus. The mRNA sequences described herein are used as vaccines and compositions for preventing or treating diseases resulting from varicella zoster virus, including shingles. Methods for formulating and preparing the vaccines are also described.

Description

mRNA COMPOSITIONS AND USES THEREOF IN VARICELLA ZOSTER VIRUS VACCINESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of U.S. Provisional Application Number 63 / 574,160, filed on April 3, 2024, the contents of which are incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed with an electronically filed Sequence Listing in XML format. The sequence listing file entitled ORB-017WOl_SL.XML was created on March 27, 2025, and is 511,122 bytes in size; the information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.BACKGROUND

[0003] Shingles, also known as herpes zoster (HZ), zoster, or zona, is a viral disease characterized by painful skin lesions (typically, a blistering rash). Shingles is caused by the varicella zoster virus (VZV).

[0004] Initial infection with VZV causes chickenpox, which generally occurs in children. Once an episode of chickenpox has resolved, the VZV is not eliminated from the body. VZV becomes latent in the nerve cell bodies and, less frequently, in non-neuronal satellite cells of dorsal root, cranial nerve, or autonomic ganglion, without causing any symptoms. Years or decades after a chickenpox infection, the virus may reactivate as shingles. The virus breaks out of nerve cell bodies and travels down nerve axons to cause viral infection of the skin in the region of the nerve (i.e., the dermatome), causing painful skin lesions. The risk of developing shingles, which increases with age and with immunosuppression due to disease and / or therapy, appears to be related to a decline in VZV-specific immunity. Shingles can cause pain, itching, or tingling of the skin followed by a painful rash of blister- like sores, fever, headache, chills, and upset stomach. This reactivation of the VZV causes more than five million cases of shingles annually. According to the Centers for Disease Control and Prevention, an estimated one million people get shingles each year in the United States, and about one out of every three people in the United States will develop shingles in their lifetime.

[0005] Shingles vaccination is the only way to protect against shingles and postherpetic neuralgia, the most common complication of shingles. It has been shown that humans vaccinated with attenuated strains of VZV have received protective immunity from VZV infections. Shingrix® is an FDA-approved vaccine indicated for prevention of herpes zoster (HZ)(shingles). Shingrix® is not indicated for prevention of primary varicella infection (chickenpox). Shingrix® is an adjuvanted recombinant VZV that unfortunately causes unwanted side effects, such as pain and swelling at injection sites, muscle pain, headache, fever and shivering. According to the Centers for Disease Control and Prevention, it is estimated that about one out of six people receiving the Shingrix® vaccine had symptoms severe enough to prevent them from performing regular activities.

[0006] There is still a strong need for improved vaccines against herpes zoster and related disorders, such as post herpetic neuralgia (PHN).SUMMARY OF THE INVENTION

[0007] The present invention provides, among other things, messenger ribonucleic acid (mRNA) molecules encoding a VZV glycoprotein E (gE) polypeptide for use as a VZV vaccine. VZV vaccine formulations, as described herein, are particularly effective in inducing cellular immune response while maintaining tolerability. This is based, in part, on the discovery of novel, codon-optimized mRNA sequences with increased expression of the VZV antigen. In some embodiments, modifications to the mRNA, including the addition of a 5’ cap, increased expression of, and response to, the VZV antigen. VZV vaccine formulations of the present invention can include, for example and without limitation, mRNA molecules encoding VZV glycoprotein E (gE) encapsulated by lipid nanoparticles (LNPs).

[0008] In some aspects, the present invention provides an mRNA molecule comprising a codon-optimized open reading frame (ORF) sequence that encodes a varicella-zoster virus (VZV) gE polypeptide. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 90% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 95% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon- optimized ORF sequence comprises a sequence at least 96% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 97% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least98% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 99% identical to any one of the sequences of SEQ ID NO: 1-95. In some embodiments, the codon-optimized ORF sequence comprises any one of the sequences of SEQ ID NO: 1-95.

[0009] In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 95% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 96% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 97% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 98% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 99% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises SEQ ID NO: 21.

[0010] In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 90% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 95% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 96% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 97% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 98% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 99% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises SEQ ID NO: 24.

[0011] In some embodiments, the mRNA further comprises a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), and / or a poly(adenine) (poly(A)) sequence. In some embodiments, the mRNA further comprises a 5’ UTR, a 3’ UTR, or a poly(A) sequence. In some embodiments, the mRNA further comprises a 5’ UTR and a 3’ UTR. In some embodiments, the mRNA further comprises a 5’ UTR and a poly(A) sequence. In some embodiments, the mRNA further comprises a 3’ UTR and a poly(A) sequence. In some embodiments, the mRNA further comprises a 5’ UTR, a 3’ UTR, and a poly(A) sequence.

[0012] In some embodiments, the mRNA comprises, from the 5’ end to the 3’ end, (a) 5’ UTR,(b) the ORF sequence, (c) the 3’ UTR, and (d) the poly(A) sequence.

[0013] In some embodiments, the mRNA further comprises a 5’ cap. In some embodiments, the mRNA comprises, from the 5’ end to the 3’ end, (a) 5’ cap, (b) 5’ UTR, (c) the ORF sequence, (d) 3’ UTR and (e) poly(A) sequence. In some embodiments, the 5’ cap is CAPO, CAP1 or CAP2. In some embodiments, the 5’ cap is CAPO. In some embodiments, the 5’ cap is CAP1 or a modified CAP1. In some embodiments, the 5’ cap is CAP2 or a modified CAP2. In some embodiments, the 5’ cap is a modified CAP1 comprising (7’ methyl)Guanosine-ppp- (2’-O-methyl)Adenosine-Guanosine (m7G-ppp-AmG). In some embodiments, the 5’ cap is a modified CAP1 comprising (7’ methyl-3’-O-methyl)Guanosine- ppp-(N-6 methyl- 2’-O-methyl) Adenosine-Guanosine ((m7-3OMe)G-ppp-(m6-2OMe)AG). In some embodiments, the 5’ cap is a modified CAP1 comprising (7’ methyl-3’-O- methyl)Guanosine-ppp- (2’-O-methyl) Adenosine-Guanosine ((m7-3OMe)G-ppp-AmG) .

[0014] In some embodiments, the mRNA comprises a sequence at least 90% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 95% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 96% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 97% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 98% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 99% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence 100% identical to any one of SEQ ID NO: 96-190.

[0015] In some embodiments, the mRNA comprises a sequence at least 90% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises a sequence at least 95% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises a sequence at least 96% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises a sequence at least 97% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises a sequence at least 98% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises a sequence at least 99% identical to SEQ ID NO: 116. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 116.

[0016] In some embodiments, the mRNA comprises a sequence at least 90% identical to SEQ ID NO: 119. In some embodiments, the mRNA comprises a sequence at least 95% identical to SEQ ID NO: 119. In some embodiments, the mRNA comprises a sequence at least 96% identical to SEQ ID NO: 119. In some embodiments, the mRNA comprises a sequence at least 97% identical to SEQ ID NO: 119. In some embodiments, the mRNA comprises a sequence at least 98% identical to SEQ ID NO: 119. In some embodiments, themRNA comprises a sequence at least 99% identical to SEQ ID NO: 119. In some embodiments, the mRNA comprises the sequence of SEQ ID NO: 119.

[0017] In some embodiments, the codon-optimized ORF encodes a VZV gE polypeptide variant. In some embodiments, the VZV gE polypeptide variant is a truncated gE polypeptide lacking the carboxy terminal domain. In some embodiments, the VZV gE polypeptide comprises a human IgG kappa (IgGK) signal peptide.

[0018] In some embodiments, the mRNA is unmodified. In some embodiments, the mRNA is chemically unmodified.

[0019] In some embodiments, the mRNA comprises at least one chemical modification. In some embodiments, the uracil nucleosides of the mRNA are chemically modified. In some embodiments, at least 80% of the uracil nucleosides are chemically modified. In some embodiments, at least 90% of the uracil nucleosides are chemically modified. In some embodiments, at least 95% of the uracil nucleosides are chemically modified. In some embodiments, at least 96% of the uracil nucleosides are chemically modified. In some embodiments, at least 97% of the uracil nucleosides are chemically modified. In some embodiments, at least 98% of the uracil nucleosides are chemically modified. In some embodiments, at least 99% of the uracil nucleosides are chemically modified. In some embodiments, at least 100% of the uracil nucleosides are chemically modified. In some embodiments, the chemical modification is in the sugar subunit of the nucleoside.

[0020] In some embodiments, the poly(A) sequence is further modified with 1-20 ribonucleic acids that are not adenosine. In some embodiments, the poly(A) sequence is modified with at least 1, at least 2, at least 3, at least 4, or at least 5 ribonucleic acids that are not adenosine. In some embodiments, the poly(A) sequence is modified with 1, 2, 3, 4, or 5 ribonucleic acids that are not adenosine.

[0021] In some embodiments, the poly(A) sequence is modified with 1, 2, 3, 4, or 5 guanosines. In some embodiments, the poly(A) sequence is modified with 1 guanosine. In some embodiments, the poly(A) sequence is modified with 2 guanosines. In some embodiments, the poly(A) sequence is modified with 3 guanosines. In some embodiments, the poly(A) sequence is modified with 4 guanosines. In some embodiments, the poly(A) sequence is modified with 5 guanosines.

[0022] In some aspects, the present invention provides mRNA formulated in a delivery vehicle. In some embodiments, the mRNA is formulated in lipid nanoparticles (LNPs). In some embodiments, the mRNA is formulated without a delivery or carrier vehicle.

[0023] In some embodiments, the LNP formulation is stored in a storage buffer. In some embodiments, the storage buffer comprises saline. In some embodiments, the storage buffer does not comprise saline.

[0024] In some embodiments, the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a polyethylene glycol (PEG)-modified lipid.

[0025] In some embodiments, the ionizable lipid comprises a compound selected from Table Cl, C2, or C3. In some embodiments, the ionizable lipid comprises 3-((((l- ethylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en-l-yl)oxy)-4-(((Z)-oct-5-en- l-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Compound 1). In some embodiments, the ionizable lipid comprises ((3-hydroxypropyl)azanediyl)bis(heptane- 7,1-diyl) bis(4,4-bis(((E)-oct-5-en-l-yl)oxy)butanoate) (Compound 37). In some embodiments, the ionizable lipid comprises ((2-hydroxyethyl)azanediyl)bis(hexane-6,l-diyl) bis(6,6-bis(hexyloxy)hexanoate) (Compound 49). In some embodiments, the ionizable lipid comprises ((2-hydroxyethyl)azanediyl)bis(heptane-7, 1-diyl) bis(4,4-bis(((Z)-oct-5-en- 1- yl)oxy)butanoate) (Compound 36). In some embodiments, the ionizable lipid comprises nonyl 8-((6-((4,4-bis(octyloxy)butanoyl)oxy)hexyl)(2-hydroxyethyl)amino)octanoate (Compound 82). In some embodiments, the ionizable lipid comprises nonyl 8-((2- hydroxyethyl)(6-((4-(((Z)-oct-5-en-l-yl)oxy)-4-(((Z)-oct-5-en-l- yl)oxy)butanoyl)oxy)hexyl)amino)octanoate (Compound 81).

[0026] In some embodiments, the ionizable lipid comprises Compound 1:(Compound 1).

[0027] In some embodiments, the ionizable lipid comprises Compound 37:(Compound 37).

[0028] In some embodiments, the ionizable lipid comprises Compound 49:(Compound 49).

[0029] In some embodiments, the ionizable lipid comprises Compound 36:(Compound 36).

[0030] In some embodiments, the ionizable lipid comprises Compound 82:(Compound 82).

[0031] In some embodiments, the ionizable lipid comprises Compound 81:(Compound 81).

[0032] In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41% cholesterol, and 1.5-2.5 % PEG-modified lipid.

[0033] In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40% cholesterol, and 2.5 % PEG-modified lipid. In some embodiments,the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.25% cholesterol, and 2.25 % PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.5% cholesterol, and 2 % PEG- modified lipid. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40.75% cholesterol, and 1.75 % PEG-modified lipid. In some embodiments, the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 41% cholesterol, and 1.5% PEG-modified lipid.

[0034] In some embodiments, the helper lipid is l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), and the PEG-modified lipid is l,2-dimyristoyl-rac-glycero-3- methoxypoly ethylene glycol-2000 (DMG-PEG2K, also known as DMG-PEG2000). In some embodiments, the helper lipid is l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and the PEG-modified lipid is DMG-PEG2K.

[0035] In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 49, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 1, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K. In some embodiments, the LNP comprises a molar ratio of 47.5 % Compound 49, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K.

[0036] In some embodiments, the present invention provides a pharmaceutical composition comprised of an mRNA described herein and a pharmaceutically acceptable carrier.In some embodiments, the present invention comprises a vector for making the mRNA described herein. In some embodiments, the vector is a non-viral DNA vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector further comprises a promoter sequence.

[0037] In some aspects, the present invention provides a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella- zoster virus (VZV) glycoprotein E (gE) polypeptide formulated in a lipid nanoparticle in an effective amount to induce in a human subject an immune response to the VZV gE polypeptide, wherein the lipid nanoparticle comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41 % cholesterol, and 1.5-2.5 % PEG-modified lipid.

[0038] In some aspects, the present invention provides a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella- zoster virus (VZV) glycoprotein E (gE) polypeptide, wherein the mRNA is formulated in a lipid nanoparticle comprising an ionizable lipid, wherein the ionizable lipid is Compound 1 or Compound 49. In some aspects, the present invention provides a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella-zoster virus (VZV) glycoprotein E (gE) polypeptide, wherein the mRNA is formulated in a lipid nanoparticle comprising an ionizable lipid comprising Compound 1. In some aspects, the present invention provides a composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella-zoster virus (VZV) glycoprotein E (gE) polypeptide, wherein the mRNA is formulated in a lipid nanoparticle comprising an ionizable lipid comprising Compound 49. In some embodiments, the LNP further comprises a helper lipid, cholesterol, and a PEG-modified lipid.

[0039] In some embodiments, the present invention provides a kit comprising the mRNA described herein. In some embodiments, the present invention provides a kit comprising the compositions described herein.

[0040] In some embodiments, the present invention provides a vaccine comprising an mRNA described herein. In some embodiments, the present invention provides a vaccine comprising the compositions described herein. In some embodiments, the vaccine further comprises an adjuvant. In some embodiments, the vaccine is a prophylactic vaccine.

[0041] In some aspects, the present invention provides method for stimulating an immune response to varicella- zoster virus (VZV) in a human subject comprising administering to the human subject an effective amount of a messenger ribonucleic acid (mRNA) comprising a codon-optimized open reading frame sequence encoding a varicella- zoster virus (VZV) glycoprotein E (gE) polypeptide, wherein the codon-optimized ORF sequence is at least 90% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is at least 95% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is at least 96% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is at least 97% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is at least 98% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is at least 99% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF sequence is 100% identical to any one of SEQ ID NO: 1-95.

[0042] In some embodiments, the method for stimulating an immune response to VZV in a human subject comprises administering to the human subject an effective amount of a mRNA comprising a sequence at least 90% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 95% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 96% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 97% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 98% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence at least 99% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA comprises a sequence 100% identical to any one of SEQ ID NO: 96-190.

[0043] In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 95% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 96% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 97% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 98% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 99% identical to SEQ ID NO: 21. In some embodiments, the codon-optimized ORF sequence comprises SEQ ID NO: 21.

[0044] In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 90% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 95% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 96% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 97% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 98% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises a sequence at least 99% identical to SEQ ID NO: 24. In some embodiments, the codon-optimized ORF sequence comprises SEQ ID NO: 24.

[0045] In some embodiments, the administration is for prophylactic treatment of a disease caused by VZV, such as shingles. In some embodiments, the immune response is a neutralizing antibody response to the glycoprotein E (gE) polypeptide encoded by the mRNAand a cellular immune response to the gE polypeptide encoded by the mRNA. In some embodiments, the immune response is a neutralizing antibody response to the gE polypeptide encoded by the mRNA or a cellular immune response to the gE polypeptide encoded by the mRNA. In some embodiments, the immune response is a neutralizing antibody response to the gE polypeptide encoded by the mRNA. In some embodiments, the immune response is a cellular immune response to the gE polypeptide encoded by the mRNA.

[0046] In some embodiments, the present invention provides a method for treating or preventing a disease caused by varicella-zoster virus (VZV) comprising administering to a human subject in need thereof the mRNA or the composition described herein. In some embodiments, the method is for treating a disease caused by VZV. In some embodiments, the method is for preventing a disease caused by VZV.

[0047] In some embodiments, the administration is to prevent primary VZV infection in the human subject. In some embodiments, the administration is to prevent herpes zoster (shingles) in an adult human subject.

[0048] In some embodiments, the mRNA is administered in a single dose. In some embodiments, the mRNA is administered in multiple doses. In some embodiments, the mRNA is administered in two doses. In some embodiments, the mRNA is administered in three doses.

[0049] In some embodiments, the mRNA is administered to the human subject via intramuscular administration. In some embodiments, the mRNA is administered to the human subject via intradermal administration. In some embodiments, the mRNA is administered to the human subject via subcutaneous administration.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The drawings are for illustration purposes only, and are not meant to be limiting.

[0051] FIG. 1 is a diagram of the gE antigen polypeptide, and shows the extracellular domain, transmembrane domain (TM), and intracellular domain. In some embodiments, exemplary mRNA VZV vaccines encode a truncated gE antigen polypeptide. For example, the gE antigen polypeptide is truncated at residue 562, 568, or 574 as marked in FIG. 1. In some embodiments, exemplary gE antigen polypeptides comprise an alanine (A) substituted in place of tyrosine (Y) at residue position 569 (Y569A mutation). Although not bound by any theory, the Y569A mutation may disrupt trans-Golgi network targeting in the intracellular domain.

[0052] FIG. 2 shows the in vitro expression of exemplary mRNA VZV gE constructs. The “Fold-over-Control” (FOC) values were calculated by dividing the geometric mean value of the VZV gE fluorescence intensity of each sample by the geometric mean value of negative control samples.

[0053] FIG. 3 shows the results from a second study testing the percent of VZV gE positive cells 24 hours, 48 hours, and 72 hours after exemplary mRNA VZV constructs were electroporated into K562 cells.

[0054] FIG. 4A - FIG 4D show the IgG titers from enzyme-linked immunosorbent assays (ELISAs) performed on serum samples collected after administration of exemplary mRNA VZV vaccines in mice. The exemplary mRNA VZV vaccines were administered intramuscularly on Day 0 and Day 21, and serum samples were collected on Day 21 and Day 42. The IgG titers include total IgG (FIG. 4A) alongside IgGl (FIG. 4B), IgG2b (FIG. 4C), and IgG2c (FIG. 4D) specific titers.

[0055] FIG. 5 A - FIG. 5B show the ratio of IgG2c titers to IgGl titers in serum samples collected after administration of exemplary mRNA VZV vaccines in mice. The exemplary mRNA VZV vaccines were administered intramuscularly on Day 0 and Day 21, and serum samples were collected on Day 21 (FIG. 5 A) and Day 42 (FIG. 5B). Higher IgG2c / IgGl ratios are indicative of T helper cell 1 (TH1) response in mice.

[0056] FIG. 6 shows the level of VZV gE-specific neutralizing antibodies titers (PRNT50 titer) in serum samples collected after administration of exemplary mRNA VZV vaccines in mice.

[0057] FIG. 7A - FIG. 7E summarize the VZV gE-specific CD4 T cell responses following administration of exemplary mRNA VZV gE vaccines in mice (Day 42). Quantification of CD4 T cell responses include interferon-gamma (IFNy)- secreting CD4 T cells (FIG. 7A), Tumor Necrosis Factor-alpha (TNFa)-secreting CD4 T cells (FIG. 7B), interleukin-2 (IL-2) secreting CD4 T cells (FIG. 7C), interleukin-4 (IL-4) secreting CD4 T cells (FIG. 7D), and interleukin-5 (IL-5) secreting CD4 T cells (FIG. 7E).

[0058] FIG. 8A - FIG. 8D summarize the VZV gE-specific CD8 T cell responses following administration of exemplary mRNA VZV gE vaccines in mice (Day 42). Quantification of CD8 T cell responses include IFNy- secreting CD8 T cells (FIG. 8A), TNF- secreting CD8 T cells (FIG. 8B), IL-2 secreting CD8 T cells (FIG. 8C), and polyfunctional CD8 T cells (FIG. 8D).

[0059] FIG. 9A - FIG. 9B show the body weight (FIG. 9A) and variation in body weight (FIG. 9B) in mice administered exemplary mRNA VZV vaccines 24 hours post-dosing (Day 1 and 22).

[0060] FIG. 10A - FIG. 10B summarize injection site reactions following administration of exemplary mRNA VZV vaccines in mice at Day 1 (FIG. 10A) and Day 22 (FIG. 10B). Increased scores represent more severe injection site reactions, while a score of 0 indicated no observed reaction.

[0061] FIG. 11 A - FIG. 1 IB show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP at Day 21 (FIG. 11 A) and Day 35 (FIG. 1 IB).

[0062] FIG. 12A - FIG. 12B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a low (1 pg) dose of exemplary mRNA VZV vaccines formulated in LNP at Day 21 (FIG. 12A) and Day 35 (FIG. 12B).

[0063] FIG. 13A - FIG. 13C summarize the CD4 T cell response to administration of a high dose of exemplary mRNA VZV vaccines formulated in LNP in mice. Quantification of CD4 T cell responses include IFNy-secreting CD4 T cells (FIG. 13A), TNF a- secreting CD4 T cells (FIG. 13B), and IL-2 secreting CD4 T cells (FIG. 13C).

[0064] FIG. 14A - FIG. 14C summarize the CD8 T cell response to administration of a high dose of exemplary mRNA VZV vaccines formulated in LNP in mice. Quantification of CD4 T cell responses include IFNy-secreting CD4 T cells (FIG. 14A), and TNFa- secreting CD4 T cells (FIG. 14B), IL-2 secreting CD4 T cells (FIG. 14C).

[0065] FIG. 15A - FIG. 15B show the body weight in mice administered a high (FIG. 15A) or low (FIG. 15B) dose of exemplary mRNA VZV vaccines formulated in LNP.

[0066] FIG. 16A - FIG. 16B summarize injection site reactions to administration of a high dose of exemplary mRNA VZV vaccines formulated in LNP in mice at Day 1 (FIG. 16 A) and Day 22 (FIG. 16B). Increased scores represent more severe injection site reactions, while a score of 0 indicated no observed reaction.

[0067] FIG. 17A - FIG. 17B summarize injection site reactions to administration of a low dose of exemplary mRNA VZV vaccines formulated in LNP in mice at Day 1 (FIG. 17A) and Day 22 (FIG. 17B). Increased scores represent more severe injection site reactions, while a score of 0 indicated no observed reaction.

[0068] FIG. 18A - FIG. 18B show the total IgG titers in response to one or two doses of exemplary mRNA VZV vaccines in mice in Experimental Groups as described in Table 4.

[0069] FIG. 19A - FIG. 19B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines with different 5’ caps in Experimental Groups as described in Table 4.

[0070] FIG. 20A - FIG. 20B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a low (1 pg) dose of exemplary mRNA VZV vaccines with different 5’ caps in Experimental Groups as described in Table 4.

[0071] FIG. 21A - FIG. 21B show the ratio of IgG2c titers to IgGl titers in serum samples collected after administration of a high (10 pg) dose of exemplary mRNA VZV vaccines with different 5’ caps in mice in Experimental Groups as described in Table 4. Higher IgG2c / IgGl ratios are indicative of T helper cell 1 (TH1) response.

[0072] FIG. 22A - FIG. 22B show the ratio of IgG2c titers to IgGl titers in serum samples collected after administration of a low (1 pg) dose of exemplary mRNA VZV vaccines with different 5’ caps in mice in Experimental Groups as described in Table 4 (FIG. 22B). Higher IgG2c / IgGl ratios are indicative of T helper cell 1 (TH1) response.

[0073] FIG. 23A - FIG. 23B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines prepared with different buffer concentrations in Experimental Groups as described in Table 4.

[0074] FIG. 24A - FIG. 24B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a low (1 pg) dose of exemplary mRNA VZV vaccines prepared with different buffer concentrations in Experimental Groups as described in Table 4.

[0075] FIG. 25 summarizes the size, polydispersity index (PDI), and encapsulation efficiency (EE%) of LNPs in different buffers after 0, 1, 3, and 5 freeze-thaw (FT) cycles.

[0076] FIG. 26 shows the percent change in the size, PDI, and mRNA encapsulation efficiency (EE%) of LNPs in various buffers after 1 FT cycle.

[0077] FIG. 27 shows the size, PDI, and EE% of LNPs with different buffer compositions after storage at 4°C for 0 days, 7 days, or 30 days.

[0078] FIG. 28 shows the size, PDI, and EE% of LNPs formulated with Compound 49 after a FT cycle.

[0079] FIG. 29 shows the effect of acidification buffer and nitrogemphosphate (N / P) ratio on the size, PDI, and EE% of LNPs formulated with Compound 1.

[0080] FIG. 30 shows the effect of acidification buffer and nitrogemphosphate (N / P) ratio on the size, PDI, and EE% of LNPs formulated with Compound 49.

[0081] FIG. 31A - FIG. 3 IB summarize the in vitro expression of VZV gE, as shown by the percent of VZV gE positive cells, after delivering exemplary mRNA VZV vaccines formulated in LNP formulations to K562 cells (FIG. 31 A), and the area under the curve (AUC) of VZV gE positive cells over time (FIG. 3 IB).

[0082] FIG. 32A - FIG. 32B summarize the in vitro expression of VZV gE, as shown by the geometric mean fluorescence intensity (gMFI), after delivering exemplary mRNA VZV vaccines formulated in LNP formulations to K562 cells (FIG. 32A), and the area under the curve (AUC) of gMFI over time (FIG. 3 IB).

[0083] FIG. 33A - FIG. 33B show the body weight (FIG. 33A) and variation in body weight (FIG. 33B) in mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP formulations.

[0084] FIG. 34A - FIG. 34B summarize injection site reactions to administration of a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP formulations in mice at Day 1 (FIG. 34A) and Day 22 (FIG. 34B). Increased scores represent more severe injection site reactions, with a score of 0 indicating no observed reaction.

[0085] FIG. 35A - FIG. 35B show the total IgG titers as measured by ELISA assays in serum samples collected from mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP formulations at Day 21 (FIG. 35A) and Day 35 (FIG. 35B).

[0086] FIG. 36A - FIG. 36C show the titers of IgGl (FIG. 36A), IgG2b (FIG. 36B), and IgG2c (FIG. 36C) as measured by ELISA assays in serum samples collected from mice administered a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP formulations.

[0087] FIG. 37 shows the ratio of IgG2c titers to IgGl titers in serum samples collected after administration of a high (10 pg) dose of exemplary mRNA VZV vaccines formulated in LNP formulations in mice. Higher IgG2c / IgGl ratios are indicative of T helper cell 1 (TH1)- response.

[0088] FIG. 38A - FIG. 38C show the percent of IFNy-secreting (FIG. 38A), IL2- secreting (FIG. 38B), and TNFa- secreting (FIG. 38C) CD4 T cells following administration of exemplary mRNA VZV vaccines formulated in LNP formulations in mice (Day 35).

[0089] FIG. 39A - FIG. 39C show the percent of IFNy-secreting (FIG. 39A), IL2- secreting (FIG. 39B), and TNFa- secreting CD8 T cells (FIG. 39C) following administration of exemplary mRNA VZV vaccines formulated in LNP formulations in mice (Day 35).

[0090] FIG. 40A - FIG. 40C show the pharmacokinetic profiles after exemplary mRNA VZV vaccines formulated in LNP formulations comprising Compound 1 and Compound 49were administered to mice. FIG. 40A shows the pharmacokinetic profile after administration in serum. FIG. 40B shows the pharmacokinetic profile after administration in liver, and FIG. 40C shows the pharmacokinetic profile after administration in injection site muscle. The pharmacokinetic profiles demonstrated that the biodegradation profiles of the mRNA VZV vaccines formulated in both LNP formulations containing Compound land Compound 49 ionizable lipids were appropriate for safe and efficacious administration.

[0091] FIG. 41A - FIG. 41D show the anti-gE VZV total IgG in serum as measured by ELISA following administration of exemplary mRNA VZV vaccines formulated in LNP formulations in mice. The anti-gE VZV total IgG in serum was quantified at Day 28 and Day 49 (FIG. 41 A), Day 53 and Day 67 (FIG. 41B), Day 81 and Day 95 (FIG. 41C), and Day 124 (FIG. 41D).

[0092] FIG. 42A - FIG. 42D show the NT50 titer (50% neutralization antibody titer) of a VZV neutralizing antibody in serum as measured by ELISA after exemplary mRNA VZV vaccines formulated in LNP was administered to mice. The NT50 in serum was quantified at Day 28 and Day 49 (FIG. 42A), Day 53 and Day 67 (FIG. 42B), Day 81 and Day 95 (FIG. 42C), and Day 124 (FIG. 42D).DEFINITIONS

[0093] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0094] Adjuvant: As used herein, the term “adjuvant” refers to agents which confer immunity by themselves. An adjuvant assists the immune system un- specifically to enhance the antigen- specific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Th 1 -based antigen specific response or vice versa. Accordingly, an adjuvant may favorably modulate cytokine expression / secretion, antigen presentation, type of immune response etc. An adjuvant or an adjuvant component in the broadest sense is typically a (e.g. pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and / or other pharmaceutically active compounds. It is to be interpreted in abroad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the context of the present invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used interchangeably.

[0095] Adaptive immune response: As used herein, the “adaptive immune response” is typically understood to be antigen- specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by “memory cells”. Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. In this context, the first step of an adaptive immune response is the activation of naive antigen- specific T cells or different immune cells able to induce an antigen- specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing. Cell types that can serve as antigen-presenting cells are inter alia dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with e.g. a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express major histocompatibility complex (MHC) molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Thl cells which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Thl cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g., of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.

[0096] Antigen: As used herein, the term “antigen” refers to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen- specific immune response, e.g., by formation of antibodies and / orantigen- specific T cells as part of an adaptive immune response. Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. In the sense of the present invention an antigen may be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, also fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigen.

[0097] Cellular immunity / cellular immune response: As used herein, the phrase “cellular immunity” relates typically to the activation of macrophages, natural killer cells (NK), antigen- specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g. by activating antigen- specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

[0098] Humoral immunity / humoral immune response: As used herein, “humoral immunity” or “humoral immune response” refers typically to antibody production and the accessory processes that may accompany it. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

[0099] Immune response: As used herein, the term “immune response” may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response). The invention relates to specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g., VZV viruses. Furthermore, this specific response can be supported by an additional unspecific reaction (innate immune response). Therefore, the invention also relates to simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient adaptive immune response.

[0100] Innate immune response: As used herein, the term “innate immune response”, also known as non-specific immune response, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. The cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g. Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL- 12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL- 26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM- CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-1 like receptor, an immuno stimulatory nucleic acid, an immunostimulatory RNA (isRNA), an antibacterial agent, or an anti-viral agent. Typically a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through, a process known as antigen presentation; and / or acting as a physical and chemical barrier to infectious agents.

[0101] Subject: As used herein, the terms “subject,” “individual,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, prognosis, treatment, or therapy is desired, particularly humans.

[0102] Treat or Treatment: As used herein, the terms “treatment,” “treat,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) increasing survival time: (b) decreasing the risk of death due to the disease; (c) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it;(d) inhibiting the disease, i.e., arresting its development (e.g., reducing the rate of disease progression); and (e) relieving the disease, e.g., causing regression of the disease.

[0103] Vaccine: As used herein, the term “vaccine” refers to a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response.

[0104] Vehicle: As used herein, the term “vehicle” refers to an agent, e.g., a carrier, that may typically be used within a pharmaceutical composition or vaccine for facilitating administering of the components of the pharmaceutical composition or vaccine to an individual.DETAILED DESCRIPTION

[0105] The present invention provides mRNA vaccines for treating herpes zoster. The present invention relates to mRNA vaccines comprising at least one mRNA molecule encoding at least one VZV antigen for use in the treatment and / or prevention of herpes zoster in an elderly patient. In some embodiments, the elderly patient preferably exhibits an age of at least 50 years. Accordingly, vaccination of the patient with mRNA vaccines described herein elicits an immune response in the patient to treat herpes zoster.A. Varicella Zoster Virus and Shingles

[0106] Varicella zoster virus (VZV) is the cause of both chickenpox and shingles. Chickenpox is associated with the primary infection by VZV. After chickenpox, VZV can permanently rest in the body’s nerves until reactivation leads to shingles. It is estimated that over 99% of Americans bom before 1980 had chickenpox and have the potential to develop shingles (also known as herpes zoster). A weakened immune system is one of the most common “triggers” for shingles and can be caused by, for example and without limitation, increased age, immune suppression, and / or stress. Around one in three people in the United States will likely develop shingles in their lifetime.

[0107] After primary infection, VZV establishes latency in ganglionic neurons. Then, when the immune system weakens, VZV reactivates by traveling anterograde towards nerve endings. There, VZV replicates in keratinocytes and epithelial cells, causing the formation of polykaryocytes. This ultimately leads to a dermatomal rash that is typically associated with shingles.

[0108] Shingles often starts with skin sensitivity, tingling, itching, and / or pain, which then develops into a rash of small, red spots that turn into blisters. Other common symptoms include fatigue, headache, and fever, but shingles can cause more serious complications. Post-herpetic neuralgia (PHN) is one such complication defined by a lasting pain sensation in the skin where the shingles rash presented. PHN occurs in around 10-18% of patients with the risk of developing PHN increasing with age. Acute complications of shingles also include, for example and without limitation, meningoencephalitis, myelitis, cranial nerve palsies, vasculopathy, gastrointestinal ulcers, pancreatitis, and hepatitis. Complications associated with shingles also lead to hospitalization in around 1-4% of patients. Increased age and / or a compromised immune system increase the likelihood of hospitalization.1. Shingles Treatment

[0109] Once shingles develops in a patient, treatment focuses on reducing pain, speeding healing, and lowering the risks of complications. Likewise, treatment for PHN is centered on relieving pain symptoms. A preferred approach to treating shingles is to prevent it from developing in the first place.

[0110] Vaccines provide a way to prime the immune system to prevent shingles from occurring. The natural immune response to VZV reactivation primarily occurs in the ganglia and in the skin. In the ganglia, local immune response is characterized by infiltration of CD4 T cells, CD8 T cells, natural killer (NK T cells), macrophages, and B cells. Similarly, immune response in the skin is associated with infiltration by CD4 T cells, CD8 T cells, NK T cells, and macrophages accompanied by increased expression of IFN, TNF, and IL-6. Effective vaccines result in the production of memory B and T cells that will more rapidly proliferate upon VZV reactivation.

[0111] Specifically, a vaccine, when injected, for example, intramuscularly, is taken up by dendritic cells and trafficked to the draining lymph node. There, MHC molecules on the dendritic cells present protein antigens from the vaccine and activate T cells. T cells then drive B cell development in the lymph node, resulting in the maturation of the antibody response. Maturation of the antibody response is associated with increases in antibody affinity and induction of different antibody isotypes.

[0112] Short-lived plasma cells will actively secrete antibodies specific for the vaccine protein, producing a rapid rise in serum antibody levels over about 2 weeks after vaccine administration. Memory B cells and CD8+ effector T cells are also produced, which are important for the elimination of infected cells. On a longer time scale, vaccines result in long-lived plasma cells that can produce antibodies for decades alongside CD8+ memory T cells that can proliferate rapidly when they encounter VZV.

[0113] VZV vaccines include, for example and without limitation, live, attenuated virus vaccines, recombinant protein vaccines, and mRNA vaccines. Live, attenuated virus vaccines carry an inherent risk of the attenuated virus reverting back to an infectious state, and use of an existing live, attenuated VZV vaccine was halted in the United States as of November 2020. Recombinant protein and mRNA vaccines do not carry any infectious material, and as such, do not carry the same risk as live, attenuated virus vaccines. Both can produce a strong immune response to a specific antigen. However, mRNA vaccines present the target antigen more naturally, as the target antigens are produced and presented by the body’s own dendritic cells. mRNA vaccines, with the appropriate formulation, also induces more of a cellular immune response than recombinant protein vaccines.B. mRNA Vaccines

[0114] In accordance with the present invention, a mRNA vaccine or composition comprises at least one mRNA molecule encoding a VZV antigen polypeptide. In some embodiments, the mRNA molecule encodes a VZV glycoprotein E (gE), or a variant thereof.1. mRNA Molecules

[0115] In some embodiments, mRNA molecules of the present invention comprise a 5’ untranslated region (UTR), an open reading frame (ORF) sequence encoding a VZV antigen polypeptide, a 3’ UTR, and / or a poly(A) sequence. In some embodiments, the mRNA molecule further comprises a 5’ cap. In some embodiments, the mRNA molecule comprises from the 5’ end to the 3’ end, 5’ UTR - ORF - 3’ UTR - poly(A) sequence. In some embodiments, the mRNA molecule comprises from the 5’ end to the 3’ end, 5’ cap - 5’ UTR - ORF - 3’ UTR - poly(A) sequence. i. Coding Sequences

[0116] The present invention provides, among other things, mRNA molecules comprising coding sequences for mRNA VZV vaccine constructs. The coding sequence, also known as open reading frame (ORF), is defined herein as the region beginning with a start codon and ending in an in-frame stop codon.

[0117] In some embodiments, the RNA molecule includes a region to initiate translation. In some embodiments, the region includes any translation initiation sequence or signal including a start codon. As a non-limiting example, the region includes a start codon. In someembodiments, the start codon is “ATG,” “ACG,” “AGG,” “ATA,” “ATT,” “CTG,” “GTG,” “TTG,” “AUG,” “AUA,” “AUU,” “CUG,” “GUG,” or “UUG”.

[0118] In some embodiments, the mRNA molecule includes a region to stop translation. This region may include any translation termination sequence or signal including a stop codon. As a non-limiting example, the region includes a stop codon. In some embodiments, the stop codon may be “TGA,” “TAA,” “TGA,” “TAG,” “UGA,” “UAA,” “UGA” or “UAG.”

[0119] In some embodiments, the regions to initiate or terminate translation independently range from 3 to 40 nucleotides, e.g., at least 4, 5-30, 10-20, 10-15, or 4-30 nucleotides in length. Additionally, in some embodiments, these regions comprise, in addition to a start and / or stop codon, one or more signal and / or restriction sequences.

[0120] In some embodiments, a masking agent is used to mask a first start codon or alternative start codon to increase the chance that translation will initiate on a start codon or alternative start codon downstream to the masked start codon or alternative start codon.

[0121] In some embodiments, the start codon is removed from the polynucleotide sequence to have the translation of the polynucleotide begin on a codon that is not the start codon. Translation of the polynucleotide begins on the codon following the removed start codon or on a downstream start codon or an alternative start codon. The polynucleotide sequence where the start codon is removed, in some embodiments, further comprises at least one masking agent for the downstream start codon and / or alternative start codons to control or attempt to control the initiation of translation, the length of the polynucleotide and / or the structure of the polynucleotide.

[0122] In some embodiments, the mRNA molecule of the present invention comprises a coding sequence. In some embodiments, the mRNA molecule of the present invention comprises an open reading frame (ORF). In some embodiments, the antigen polypeptide comprises an amino acid sequence selected from Table A, or variants thereof.Table A. Exemplary sequences of mRNA VZV vaccines.

[0123] In some embodiments, the mRNA molecule encodes a wild-type VZV gE antigen comprising an amino acid sequence of SEQ ID NO: 199, including the underlined signal peptide.MGTVNKPWGVLMGFGI I TGTLRI TNPVRASVLRYDDFH I DE DKL D TN S VYEP Y YH SDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERL MQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRL IEVSVEENHPFTLRAPIQRI YGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQ DVWDVDCAENTKEDQLAEI SYRFQGKKEADQPWIWNTSTLFDELELDPPEIEPGV LKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFH MWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYH PNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGI SHMEPSFGLIL HDGGTTLKFVDTPESLSGLYVFWYFNGHVEAVAYTWSTVDHFVNAIEERGFPPTA GQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAWLLCLVIFLICTAKRMRVKAYR VDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR( SEQ ID NO : 199 )

[0124] In some embodiments, the mRNA molecule encodes a truncated VZV gE antigen.In some embodiments, the mRNA molecule encodes a mutated VZV gE antigen.

[0125] In some embodiments, the mRNA molecule encodes a VZV gE polypeptide variant. In some embodiments, the VZV gE polypeptide variant is truncated. In some embodiments, the truncated VZV gE polypeptide variant lacks the carboxy terminal domain.

[0126] In some embodiments, the truncated VZV gE antigen comprises residues 1-561 of the wild-type VZV antigen. In some embodiments, the truncated VZV gE antigen comprises residues 1-567 of the wild-type VZV antigen. In some embodiments, the truncated VZV GE antigen comprises residues 1-573 residues of the wild-type VZV antigen. In some embodiments, the mutated VZV antigen comprises a Y569A mutation relative to the wildtype VZV antigen. In some embodiments, the truncated VZV antigen further comprises aY569A mutation relative to the wild-type VZV antigen.

[0127] In some embodiments, the mRNA molecule encodes, in part, a signal peptide. In some embodiments, the mRNA molecule encodes a signal peptide derived from VZV gE. In some embodiments, the mRNA molecule encodes a signal peptide derived from IgGK. Insome embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 196:MGTVNKPWGVLMGFGI I TGTLRI TNPVRA ( SEQ ID NO : 196 )

[0128] In some embodiments, the signal peptide comprises an amino acid sequence of SEQ ID NO: 198:METPAQLLFLLLLWLPDTTG ( SEQ ID NO : 198 ) ii. Codon Optimization

[0129] The mRNA molecules of the present invention, their regions or parts or subregions may be codon-optimized. In some embodiments, the coding nucleic acid sequences are codon-optimized. In some embodiments, the coding sequences of the mRNA molecules are codon-optimized. In some embodiments, the ORF sequences of the mRNA molecules are codon-optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include, but are not limited to, match codon frequencies in target and host organisms to ensure proper folding, alter GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide.

[0130] In some embodiments, a codon-optimized sequence may be one in which codons in a polynucleotide encoding a polypeptide have been substituted to increase the expression, stability and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to one or more of the following: (i) variation of codon biases between two or more organisms or genes or synthetically constructed bias tables, (ii) variation in the degree of codon bias within an organism, gene, or set of genes, (iii) systematic variation of codons including context, (iv) variation of codons according to their decoding tRNAs, (v) variation of codons according to GC %, either overall or in one position of the triplet, (vi) variation in degree of similarity to a reference sequence for example a naturally occurring sequence, (vii) variation in the codon frequency cutoff, (viii) structural properties of mRNAs transcribed from the DNA sequence, (ix) prior knowledge about the function of the DNAsequences upon which design of the codon substitution set is to be based, and / or (x) systematic variation of codon sets for each amino acid.

[0131] Codon optimization tools, algorithms and services are known in the art; nonlimiting examples include, but are not limited to, software from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.), and / or proprietary methods.

[0132] In some embodiments, the coding sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table B.Table B. Codon Options.

[0133] In some embodiments, the coding sequences were codon-optimized to increase expression of VZV gE. In some embodiments, the coding sequences were codon-optimized to increase immune response to VZV gE. In some embodiments, the coding sequences were codon-optimized to increase mRNA stability. In some embodiments, the coding sequences were codon-optimized to increase mRNA half-life.

[0134] In some embodiments, the coding sequences were optimized for human codon usage. In some embodiments, the coding sequences were optimized for GC content. In someembodiment, the coding sequences were optimized for human codon usage and GC content. In some embodiments, the coding sequences were optimized for structure stability. In some embodiments, the coding sequences were optimized for human codon usage and structure stability. In some embodiments, the coding sequences were optimized for human codon usage, GC content, and structure stability. iii. Exemplary Coding Sequences

[0135] In some embodiments, the ORF comprises a sequence identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 90% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 95% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 96% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 97% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 98% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises a sequence at least 99% identical to any one of SEQ ID NO: 1-95. In some embodiments, the ORF comprises the sequence of any one of SEQ ID NO: 1-95.

[0136] In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 4. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 8. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ IDNO: 9. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 12. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 13. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 14. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 19. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 27. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 29. In some embodiments, the ORF comprises a sequence atleast 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 30. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 33. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 34. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 35. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 36. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 38. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 39. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 40. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 41. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 42. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 43. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 44. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 46. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 47. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 48. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 49. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to SEQ ID NO: 50. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 52. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 53. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 54. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 55. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 56. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 57. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 58. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 59. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 60. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 61. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 62. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 63. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 64. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 65. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 66. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 67. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 68. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 69. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 70. In some embodiments,the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 71. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 72. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 73. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 74. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 75. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 76. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 77. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 78. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 79. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 80. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 81. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 82. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 83. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 84. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 85. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 86. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 87. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 88. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 89. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 90. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 91. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 92. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 93. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% , or 100% identical to SEQ ID NO: 94. In some embodiments, the ORF comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 95.

[0137] In some embodiments, the ORF comprises a sequence at least 90% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 95% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 96% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 97% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 98% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence at least 99% identical to SEQ ID NO: 21. In some embodiments, the ORF comprises a sequence identical to SEQ ID NO: 21.

[0138] In some embodiments, the ORF comprises a sequence at least 90% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 95% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 96% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 97% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 98% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence at least 99% identical to SEQ ID NO: 24. In some embodiments, the ORF comprises a sequence identical to SEQ ID NO: 24.

[0139] In addition to the coding sequences, the mRNA molecules of the present disclosure comprise regions that are partially or substantially not translatable, e.g., having a noncoding region. Such noncoding regions are different from the non-coding functional sequences and are located in any region of the mRNA molecule. The non-coding regions include but are not limited to the linker, the spacer and / or the flanking regions. In some embodiments, the noncoding regions are located in more than one region of the mRNA molecule. iv. Untranslated regions (UTRs)

[0140] In some embodiments, the mRNA molecule comprises at least one untranslated region (UTR), such as a 5’ UTR and / or 3’ UTR. Untranslated regions (UTRs) of a gene aretranscribed but not translated. The 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3 'UTR starts immediately following the stop codon and continues until the transcription termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides, primary constructs and / or mRNA molecules of the present invention to enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in unwanted tissues.

[0141] In some embodiments, the mRNA molecule comprises a 5’ UTR. In some embodiments, the 5’UTR is a natural 5’ UTR. Natural 5' UTRs bear features which play roles in translation initiation. The 5'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5 '-Terminal Oligopyrimidine Tract. The 5’ UTR may also harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of eukaryotic genes. Kozak sequences have the consensus GCCRCCAUG, where R is a purine (adenosine or guanosine) three bases upstream of the start codon (AUG). In some embodiments, the 5’UTR is an engineered 5’UTR. By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the mRNA molecules of the present disclosure.

[0142] Other non-UTR sequences may be incorporated into the 5' or 3' UTR. For example, and without limitation, sequences known to bind RNA Binding Proteins (RBPs). Incorporation of RBP sequences may increase protein production.

[0143] In some embodiments, the mRNA molecule comprises a 5' spacer. In some embodiments, the 5' spacer sequence is about 10-100 nucleotides in length.

[0144] The 5' UTR may be post-transcriptionally modified, for example by addition of a 5'-cap. In some embodiments, the 5' UTR corresponds to the sequence of a naturally occurring mRNA which is located between the 5' cap and the start codon. Preferably, the 5' UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5' cap, preferably from the nucleotide located immediately 3' to the 5' cap, to a nucleotide located 5' to the start codon of the protein coding region, preferably to the nucleotide located immediately 5' to the start codon of the protein coding region. The nucleotide located immediately 3' to the 5' cap of a mature mRNA typically corresponds to the transcriptional start site. The term “corresponds to” means that the 5' UTR sequence may be an RNAsequence, such as in the mRNA sequence used for defining the 5' UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 5' UTR of a gene”, such as “a 5' UTR of a TOP gene”, is the sequence which corresponds to the 5' UTR of the mature mRNA derived from this gene, i.e., the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “5' UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5' UTR.

[0145] Exemplary 5’ UTR sequences include SEQ ID NO: 191 and SEQ ID NO: 193.

[0146] In some embodiments, the mRNA molecule comprises a 5’ UTR sequence comprising GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 191).

[0147] In some embodiments, the mRNA molecule comprises a 5’ UTR sequence comprising AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 193).

[0148] In some embodiments, the mRNA molecule provided herein comprises 3' UTR. A 3' UTR is typically the part of an mRNA which is located between the protein coding region (i.e., the open reading frame) and the poly(A) sequence of the mRNA. A 3' UTR of the mRNA is not translated into an amino acid sequence. The 3' UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process.

[0149] In the context of the present invention, a 3' UTR corresponds to the sequence of a mature mRNA which is located 3' to the stop codon of the protein coding region, preferably immediately 3' to the stop codon of the protein coding region, and which extends to the 5'- side of the poly (A) sequence, preferably to the nucleotide immediately 5' to the poly (A) sequence. The term “corresponds to” means that the 3' UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3' UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 3' UTR of a gene”, such as “a 3' UTR of an albumin gene”, is the sequence which corresponds to the 3' UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “3' UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3' UTR.

[0150] 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates ofturnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of the mRNA molecule of the present disclosure.

[0151] In some embodiments, the 3' UTR may be derived from human beta-globin, human alpha-globin, xenopus beta-globin, xenopus alpha-globin. In some embodiments, the 3’ UTR is an engineered UTR sequence.

[0152] Exemplary 3’ UTR sequences include SEQ ID NO: 192 and SEQ ID NO: 194.

[0153] In some embodiments, the mRNA molecule comprises a 3’ UTR sequence comprising GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC (SEQ ID NO: 192).

[0154] In some embodiments, the mRNA molecule comprises a 3’ UTR sequence comprising GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCU CCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGG CGGC (SEQ ID NO: 194). v. Poly (A) sequence

[0155] In some embodiments, the mRNA molecule comprises a poly A sequence (also called as poly (A) tail).

[0156] A poly(A) sequence is typically a long sequence of adenosine nucleotides of up to about 500 adenosine nucleotides, e.g., from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides, added to the 3'-end of an RNA. Moreover, poly(A) sequences, or poly(A) tails may be generated in vitro by enzymatic polyadenylation of the RNA, e.g., using Poly(A)polymerases derived from E. coli or yeast.

[0157] In some embodiments, the length of a poly(A) tail is greater than 30 nucleotides in length. In some embodiments, the poly A tail is greater than 25 nucleotides in length (e.g., at least or greater than about 25, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, and 3000 nucleotides). In some embodiments, the poly A tail includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to1.500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to2.500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[0158] In some embodiments, the length of the poly A tail is relative to the length of the overall mRNA molecule. In some embodiments, the length is relative to the length of the coding region or another particular feature or region.

[0159] In some embodiments, the poly(A) tail is further modified to enhance protein production. In some embodiments, the poly(A) tail is further modified to improve stability. In some embodiments, the poly(A) tail is further modified with 1-20 (e.g., 1-10, 2-10, 10-20, 1- 5, 1-3, 3-5) ribonucleic acids that are not adenosine. In some embodiments, the poly(A) tail is further modified with 1, 2, 3, 4, or 5 ribonucleic acids that are not adenosine.

[0160] In some embodiments, the poly(A) tail is further modified with 1, 2, 3, 4, or 5 guanosines. In some embodiments, the poly(A) tail is further modified with 1 guanosine. In some embodiments, the poly(A) tail is further modified with 2 guanosines. In some embodiments, the poly(A) tail is further modified with 3 guanosines. In some embodiments, the poly(A) tail is further modified with 4 guanosines. In some embodiments, the poly(A) tail is further modified with 5 guanosines.

[0161] In some embodiments, the poly(A) tail is further modified with 1, 2, 3, 4, or 5 cytosines. In some embodiments, the poly(A) tail is further modified with 1 cytosine. In some embodiments, the poly(A) tail is further modified with 2 cytosines. In some embodiments, the poly(A) tail is further modified with 3 cytosines. In some embodiments, the poly(A) tail isfurther modified with 4 cytosines. In some embodiments, the poly(A) tail is further modified with 5 cytosines.

[0162] In some embodiments, the poly(A) tail is further modified with 1, 2, 3, 4, or 5 uracils. In some embodiments, the poly(A) tail is further modified with 1 uracil. In some embodiments, the poly(A) tail is further modified with 2 uracils. In some embodiments, the poly(A) tail is further modified with 3 uracils. In some embodiments, the poly(A) tail is further modified with 4 uracils. In some embodiments, the poly(A) tail is further modified with 5 uracils. vi. 5’ Capping

[0163] In some embodiments, a mRNA may further be modified at 5’ end with a 5' cap structure. The 5’ cap structure of a linear RNA, or mRNA, is required for mRNA translation and stability in the cytoplasm. The 5’ cap structure binds to Eukaryotic translation initiation factor 4e (eIF4e) which, in turn, recruits the remaining translation initiation complex. Additionally, the 5’ cap protects the mRNA from 5 ’-3’ exonuclease digestion by Xml.

[0164] A 5' cap is typically a modified nucleotide, particularly a guanosine nucleotide containing a methyl group at the 7 position, added to the 5 '-end of an mRNA molecule through a 5 '-5 '-triphosphate linkage, denoted as m7GpppN, where N is the first transcribed base of the mRNA). In some embodiments, N is alanine. In some embodiments, N is guanosine. In some embodiments, N is uracil. In some embodiments, N is cytosine. In some embodiments, N is an unmodified nucleotide. In some embodiments, N is a modified nucleotide.

[0165] Synthetic mRNA can be capped enzymatically (e.g., with vaccinia capping enzyme) or through co-transcription with a di- or tri-nucleotide cap analog (e.g., m7GpppApG). These modified 5' cap structures may be used in the context of the present invention to modify the mRNA sequence of the inventive composition. Further modified 5' cap structures which may be used in the context of the present invention include but are not limited to CAPO, CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppNm), CAP2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppNmNm), CAP3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppNmNmNm), CAP4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppNmNmNmNm), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, Nl-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0166] In some embodiments, a cap analogue can be a non-polymerizable di- or trinucleotide that has cap functionality in that it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5 '-end of the RNA molecule. Non-polymerizable means that the cap analogue will be incorporated only at the 5'- terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3 '-direction by a template-dependent RNA polymerase. Cap analogues include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated cap analogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G), or anti reverse cap analogues (e.g., ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10): 1486-495). Further cap analogues have been described previously (U.S. Pat. No. 7,074,596, W02008 / 016473, WO2008 / 157688, WO2009 / 149253, WO2011 / 015347, and WO2013 / 059475).

[0167] In some embodiments, the 5’ cap comprises m7GpppAG, m7GpppGG, 3’0Me,m7Gppp-m6AG, or 3’0Me,m7Gppp-AG. In some embodiments, the 5’ cap comprises m7GpppAG. In some embodiments, the 5’ cap comprises m7GpppGG. In some embodiments, the 5’ cap comprises 3’0Me,m7Gppp-m6AG. In some embodiments, the 5’ cap comprises 3’0Me,m7Gppp-AG. vii. Modified Nucleotides

[0168] In some embodiments, the mRNA molecule comprises one or more modified nucleotides. In some embodiments, the mRNA molecule is unmodified.

[0169] In some embodiments, the mRNA molecules of the present invention may include one, two, three, or more modifications. In some embodiments, the modified nucleotides are located in coding region(s). In some embodiments, the modified nucleotides are in the untranslated region(s).

[0170] In some embodiments, the modifications stabilize the mRNA molecule and enhance resistance to degradation as compared to unmodified nucleotides. In some embodiments, modified nucleotides enhance biological functions of nucleic acid molecules, for example, increase binding to a RNA binding protein or increasing translation.

[0171] In some embodiments, the modified nucleotide is one or more of Nl- methylpseudouridine, 5-methoxyuridine, N6-methyladenosine, pseudouridine or 5- methylcytosine.

[0172] In some embodiments, the modified nucleotide is Nl-methylpseudouridine. In some embodiments, the modified nucleotide is 5-methoxyuridine. In some embodiments, the modified nucleotide is N6-methyladenosine. In some embodiments, the modified nucleotide is pseudouridine. In some embodiments, the modified nucleotide is 5-methylcytosine.

[0173] In some embodiments, the modified nucleotide is 100%. In some embodiments, the modified nucleotide is less than 50%. In some embodiments, the modified nucleotide is less than 20%. In some embodiments, the modified nucleotide is less than 10%.

[0174] The mRNA molecules of the present disclosure may contain from about 0% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, T / U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 85% to 95%, from 85% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).

[0175] In some embodiments, the mRNA molecules are 100% modified. In some embodiments, the mRNA molecules are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 75% modified, e.g., at least 75% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 20% modified, e.g., at least 20% of the nucleotides are modified. In some embodiments, the mRNA molecules are at least 10% modified, e.g., at least 10% of the nucleotides are modified. It is to be understood that since a nucleotide (sugar, base and phosphate moiety, e.g., linkage) may each be modified, any modification to any portion of a nucleotide, or nucleoside, will constitute a modification.

[0176] In some embodiments, the modifications are structural modifications and / or chemical modifications. In some embodiments, the chemical modification is a nucleotideand / or nucleoside modification including a nucleobase modification and / or a sugar modification, and a backbone linkage modification (i.e., the intemucleoside linkage, e.g., a linking phosphate, a phosphodiester linkage, and a phosphodiester backbone). In some embodiments, the structural modification includes a secondary and / or tertiary structural modification.

[0177] In some embodiments, modifications include modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof.

[0178] In some embodiments, one, two, or more (optionally different) nucleoside or nucleotide modifications may be incorporated to the circular polynucleotides of the present disclosure.

[0179] In some embodiments, the mRNA molecule comprises at least one modification described herein. In other embodiments, the mRNA molecules comprise two, three, four, or more (optionally different) chemical modifications described herein. The modifications may be combinations of nucleobase (purine and / or pyrimidine), sugar and backbone (internucleoside) linkage modifications. The modifications may be located on one or more nucleotides of the circular polynucleotide. In some embodiments, all the nucleotides of the mRNA molecule are chemically modified. In some embodiments, all the nucleotides of the nucleic acid sequence with a biological function are chemically modified.

[0180] In some embodiments, the mRNA molecules are at least 10% modified in only one component of the nucleotide, with such component being the nucleobase, sugar, or linkage between nucleosides. For example, modifications may be made to at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleobases, sugars, or linkages of a polynucleotide described herein.

[0181] In some embodiments, the mRNA molecules are designed with a patterned array of sugar, nucleobase or linkage modifications.

[0182] In some embodiments, the mRNA molecules comprise modifications to maximize stability.

[0183] In other embodiments, the mRNA molecules comprise modifications to decrease stability.

[0184] In some embodiments, the modified nucleosides and nucleotides include a modified nucleobase. Examples of nucleobases in RNA include, but are not limited to, adenosine(A), guanosine(G), cytosine(C), and uracil(U). Examples of nucleobases in DNA include, but are not limited to, adenine(A), guanine(G), cytosine(C), and thymine(T).

[0185] In some embodiments, the modified nucleobase is a modified uracil(U). Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin-4- one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4- thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5- aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine (I5U) or 5-bromo-uridine (br5U)), 3- methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5 -carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U),1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxy thy mine), 1- methylpseudouridine (nTy), 5-methyl-2-thio-uridine (m5s2U), pseudouracil (y), l-methyl-4- thio-pseudouridine (m1, 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3y),2-thio- 1 -methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl- dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio- pseudouridine, N1 -methyl-pseudouridine (also known as 1 -methylpseudouridine (m, 3- (3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ym), 2-thio-2'-O- methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O- methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)- 2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F- uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E- propenylamino)uridine.

[0186] In some embodiments, the modified nucleobase is a modified cytosine(C). Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine,6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5- formyl-cytidine (FC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5 -hydroxy methyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl- cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (FCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0187] In some embodiments, the modified nucleobase is a modified adenosine(A). Exemplary nucleobases and nucleosides having a modified adenosine include 2-amino- purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo- purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza- adenosine, 7-deaza-8-aza- adenosine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine,7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (m1A), 2- methyl-adenosine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6- threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2- methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6- hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl- adenosine, 2-methylthio-adenosine, 2-methoxy-adenosine, a-thio-adenosine, 2'-O-methyl- adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), l,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2- amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F- adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0188] In some embodiments, the modified nucleobase is a modified guanosine(G). Exemplary nucleobases and nucleosides having a modified guanosine include inosine (I), 1- methyl-inosine (m1!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (02yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuo sine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7 -deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (m'G), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2’2’7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl- guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl- guanosine (nAGm), l-methyl-2'-O-methyl-guanosine (m 'Gm), N2,7-dimethyl-2'-O-methyl- guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (m1hn), and 2'-O- ribosylguanosine (phosphate) (Gr(p)).

[0189] In some embodiments, the nucleobase of the nucleotide is independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase and / or analog is each independently selected from adenosine, cytosine, guanosine, uracil, naturally-occurring and synthetic derivatives of a base, including but not limited to pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenosine, 6-methyl and other alkyl derivatives of adenosine and guanosine, 2-propyl and other alkyl derivatives of adenosine and guanosine, 2- thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenosines and guanosines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanosine and 7-methyladenosine, 8-azaguanosine and 8-azaadenosine, deazaguanosine, 7-deazaguanosine, 3-deazaguanosine, deazaadenosine, 7-deazaadenosine, 3- deazaadenosine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] 1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.

[0190] In some embodiments, the mRNA molecule comprises a nucleoside modification. In some embodiments, one or more atoms of a pyrimidine nucleobase is replaced or substituted, for example, with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), optionally substituted or halo (e.g., chloro or fluoro) atoms or groups.

[0191] In some embodiments, uracil nucleosides of the mRNA molecule of the present disclosure are all modified. In some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% of the uracil nucleosides of the mRNA molecule are modified. In some embodiments, the uracil nucleosides of the mRNA molecule comprise modifications in the sugar subunit.

[0192] In some embodiments, the guanosine nucleosides of the mRNA molecule of the present disclosure are all modified. In some embodiments, the cytosine nucleosides of the mRNA molecule of the present disclosure are all modified. In some embodiments, the thymine nucleosides of mRNA molecule of the present disclosure are all modified. In some embodiments, the adenosine nucleosides of mRNA molecule of the present disclosure are all modified. In some embodiments, the modifications to each nucleobase are the same. In some embodiments, the modifications to each nucleobase are different.

[0193] In some embodiments, modifications of the modified nucleosides and nucleotides are present in the sugar subunit. In some embodiments, the mRNA molecule described herein comprise at least one sugar modification. Generally, mRNA includes the sugar subunit: ribose, which is a 5-membered ring having an oxygen. In some embodiments, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'OH-position include, but are not limited to, H, halo, optionally substituted Ci-6 alkyl; optionally substituted Ci-6 alkoxy; optionally substituted Ce-io aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted Ce-io aryloxy; optionally substituted Ce-io aryl-Ci-6 alkoxy, optionally substituted Ci-i2 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG)-O(CH2CH2O)nCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); and “locked” nucleic acids (LNA) in which the 2'-hydroxyl is connected by a C1-6 alkylene or C1-6 heteroalkylene bridge to the 4’ -carbon of the same ribose sugar, whereexemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0194] Other exemplary sugar modifications include replacement of the oxygen(O) in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'— >2')) , and peptide nucleic acid (PNA, where 2-amino-ethyl- glycine linkages replace the ribose and phosphodiester backbone).

[0195] In some embodiments, the sugar subunit contains one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. In some embodiments, mRNA molecules as described herein, include nucleotides containing, e.g., arabinose, as the sugar.

[0196] In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the mRNA molecules is substituted with -O- methoxyethyl, referred to as 2’-0Me. In some embodiments, at least one of the 2' positions of the sugar (OH in RNA or H in DNA) of a nucleotide of the polynucleotides is substituted with -F, referred to as 2’-F. In some embodiments, the sugar modification is one or more locked nucleic acids (LNAs). In some embodiments, the polynucleotides are fully 2’-MOE-sugar modified.

[0197] In some embodiments, one or more modifications are present in the intemucleoside linkage (the linking phosphate or the phosphodiester linkage or the phosphodiester backbone). In the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably.

[0198] In some embodiments, backbone phosphate groups are modified by replacing one or more of the oxygen atoms with a different substituent. In some embodiments, modified nucleosides and nucleotides include replacement of an unmodified phosphate moiety with another intemucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonates phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioateshave both non-linking oxygens replaced by sulfur. The phosphate linker is also modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0199] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides through unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked polynucleotide molecules are expected to also reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0200] In some embodiments, the mRNA molecules of the present disclosure comprise at least one phosphorothioate linkage, methylphosphonate linkage between nucleotides, 5'-(E)- vinylphosphonate (5'-E-VP), a phosphate mimic, as a modification.

[0201] In some embodiments, the intemucleoside linkages of the mRNA molecules may be partially or fully modified.

[0202] In some embodiments, modified nucleotides incorporated in the mRNA molecules include, for example, 2’-O-Methyl-modified or 2’-O-Methoxyethyl-modified nucleotides (2’- OMe and 2’-M0E modifications, respectively), an alpha-thio-nucleoside (e.g., 5'-O-(l- thiophosphate)-adenosine, 5'-O-(l-thiophosphate)-cytidine (a-thio-cytidine), 5'-O-(l- thiophosphate)-guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)- pseudouridine.

[0203] Additional modifications to mRNA molecules of the present disclosure include, for example, modification or deletion of nucleotides (or codons) encoding one or more N-linked glycosylation site in a translated polypeptide.

[0204] In some embodiments, different sugar modifications, nucleobase modifications, and / or intemucleoside linkages (e.g., backbone structures) are introduced at various positions in a polynucleotide described herein. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased.

[0205] In some embodiments, the one or more modified nucleotides is a 2' O-methyl or a phosphorothioate modified nucleotide. Accordingly, in some embodiments, the one or more modified nucleotides comprises a 2' O-methyl modification. In some embodiments, the one or more modified nucleotides comprises a phosphorothioate modification.

[0206] In some embodiments, the one or more modified nucleotides is selected from 2'-O- methyl 3 '-phosphorothioate, 2'O-methyl, 2'-ribo 3 '-phosphorothioate, 2'-fluro, 2’-O-methoxyethyl morpholino (PMO), locked nucleic acid (LNA), deoxy, or 5' phosphate modified nucleotide. Accordingly, in some embodiments, the one or more modified nucleotides is a 2'-O-methyl 3'-phosphorothioate. In some embodiments, the one or more modified nucleotides is a 2'-O-methyl nucleotide. In some embodiments, the one or more modified nucleotides is a 2'-ribo 3'-phosphorothioate. In some embodiments, the one or more modified nucleotides is a 2'-fluro nucleotide. In some embodiments, the one or more modified nucleotides is a locked nucleic acid (LNA). In some embodiments, the one or more modifications comprises a 2’-O-methoxyethyl morpholino (PMO). In some embodiments, the one or more modifications comprises a deoxy modification. In some embodiments, the one or more modifications comprises a 5' phosphate modification.

[0207] Various modified RNA bases are known in the art and include for example, 2'-O- methoxy-ethyl bases (2'-MOE) such as 2-MethoxyEthoxy A, 2-MethoxyEthoxy MeC, 2- MethoxyEthoxy G, 2-MethoxyEthoxy T. Other modified bases include for example, 2'-O- Methyl RNA bases, and fluoro bases. Various fluoro bases are known, and include for example, Fluoro C, Fluoro U, Fluoro A, Fluoro G bases. Various 2'OMethyl modifications can also be used with the methods described herein. For example, the following RNA comprising one or more of the following 2'OMethyl modifications can be used with the methods described: 2'-OMe-5-Methyl-rC, 2'-OMe-rT, 2'-OMe-rI, 2'-OMe-2-Amino-rA, Aminolinker-C6-rC, Aminolinker-C6-rU, 2'-OMe-5-Br-rU, 2'-OMe-5-I-rU, 2-OMe-7-Deaza- rG.

[0208] In some embodiments, the mRNA molecule comprises one or more of the following modifications: phosphorothioates, 2'0-methyls, 2' fluoro (2'F), DNA. In some embodiments, the mRNA molecule comprises 2'OMe modifications at the 3' and 5 '-ends. In some embodiments, the mRNA molecule comprises one or more of the following modifications: 2' -O-2-Methoxyethyl (MOE), locked nucleic acids, bridged nucleic acids, unlocked nucleic acids, peptide nucleic acids, morpholino nucleic acids. In some embodiments, the mRNA molecule comprises one or more of the following base modifications: 2,6-diaminopurine, 2-aminopurine, pseudouracil, Nl-methyl-psuedouracil, 5' methyl cytosine, N6-methyladenosine, 2'pyrimidinone (zebularine), thymine. Other modified bases include for example, 2- Aminopurine, 5-Bromo dU, deoxyUridine, 2,6-Diaminopurine (2-Amino-dA), Dideoxy-C, deoxyinosine, Hydroxymethyl dC, Inverted dT, Iso-dG, Iso-dC, Inverted Dideoxy-T, 5-Methyl dC, 5-Methyl dC, 5-Nitroindole, Super T®, 2'-F-r(C,U), 2'- NH2-r(C,U), 2,2'-Anhydro-U, 3'-Desoxy-r(A,C,G,U), 3'-O-Methyl-r(A,C,G,U), rT, rl, 5-Methyl-rC, 2-Amino-rA, rSpacer (Abasic), 7-Deaza-rG, 7-Deaza-rA, 8-Oxo-rG, 5- Halogenated-rU, N-Alkylated-rN.

[0209] In some embodiments, other chemically modified RNA is used herein. For example, the mRNA molecule can comprise a modified base such as, for example, 5', Int, 3' Azide (NHS Ester); 5' Hexynyl; 5', Int, 3' 5-Octadiynyl dU; 5', Int Biotin (Azide); 5', Int 6- FAM (Azide); and 5', Int 5-TAMRA (Azide). Other examples of RNA nucleotide modifications that can be used with the methods described herein include for example phosphorylation modifications, such as 5 '-phosphorylation and 3 '-phosphorylation. The mRNA molecule can also have one or more of the following modifications: an amino modification, biotinylation, thiol modification, alkyne modifier, adenylation, Azide (NHS Ester), Cholesterol-TEG, and Digoxigenin (NHS Ester).2. mRNA Production

[0210] In some embodiments, the mRNA molecule is synthetic RNA. In some embodiments, the mRNA molecule is chemically synthesized. In some embodiments, RNA synthesis is carried out in synthesizer machines using nucleotide triphosphate derivatives known as phosphoramidites, which are building blocks of linear oligonucleotides. Nucleoside phosphoramidites use inert substituents to protect reactive moieties such as hydroxyl and amino groups from undesirable reactions and promote phosphodiester bond formation leading to greater homogenous yields. Once synthesis is complete, these groups are removed to generate RNA oligonucleotides of high purity. The mRNA described herein can be purified by methods commonly known in the art.

[0211] In some embodiments, the mRNA molecule is in vitro transcribed RNA. An in vitro transcription (IVT) reaction typically comprises a double-stranded DNA (dsDNA) template, ribonucleotide triphosphates, and a DNA-dependent RNA polymerase. In some embodiments, the DNA-dependent RNA polymerase is derived from bacteriophage. In some embodiments, the DNA-dependent RNA polymerase is a T7 RNA polymerase, SP6 RNA polymerase, or T3 RNA polymerase. In some aspects, the present invention provides DNA templates for the RNA molecules described herein.

[0212] The DNA template contains a promoter sequence to which the polymerase binds and catalyzes downstream transcription. In some embodiments, the promoter is about 20-40 nucleotides (nt) long. In some embodiments, the DNA template is a double-stranded PCR product. In some embodiments, the DNA template is a linearized plasmid containing a promoter upstream of the DNA sequence to be transcribed.

[0213] In some embodiments, viral vectors are used to package the constructs for producing the mRNA molecules described herein. In some embodiments, AAV vectors are used to construct the mRNA molecules. In other embodiments, non- viral vectors such as plasmids, cosmids and artificial chromosomes are used to construct the mRNA molecules.3. Exemplary mRNA molecules used for vaccines

[0214] In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NO: 96-190 (Table 9). In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 96. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 97. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 98. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 99. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 100. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 101. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 102. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 103. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 104. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 105. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 106. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 107. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 108. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 109. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 110. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 111. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 112. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 113. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 114. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 115. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 116. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 117. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 118. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 119. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 120. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 121. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 122. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 123. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 124. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 125. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 126. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 127. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 128. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 129. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 130. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 131. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 132. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 133. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 134. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 135. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 136. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 137. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 138. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 139. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 140. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 141. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 142. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 143. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 144. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 145. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 146. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 147. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 148. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 149. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 150. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 151. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 152. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 153. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 154. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 155. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 156. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 157. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 158. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 159. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 160. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 161. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 162. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 163. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 164. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 165. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 166. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 167. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 169. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 170. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 171. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 172. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 173. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 174. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 175. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to SEQ ID NO: 176. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 177. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 178. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 179. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 180. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 181. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 182. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 183. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 184. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 185. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 186. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 187. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 188. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 189. In some embodiments, an mRNA molecule for VZV vaccine comprises a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 190.

[0215] In some embodiments, the mRNA molecule comprises a sequence at least 90% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA molecule comprises a sequence at least 95% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA molecule comprises a sequence at least 96% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA molecule comprises a sequence atleast 97% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA molecule comprises a sequence at least 98% identical to any one of SEQ ID NO: 96-190. In some embodiments, the mRNA molecule comprises a sequence at least 99% identical to any one of SEQ ID NO: 96-190.

[0216] In some embodiments, the mRNA molecule comprises a sequence at least 90% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence at least 95% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence at least 96% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence at least 97% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence at least 98% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence at least 99% identical to SEQ ID NO: 116. In some embodiments, the mRNA molecule comprises a sequence identical to SEQ ID NO: 116.

[0217] In some embodiments, the mRNA molecule comprises a sequence at least 90% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence at least 95% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence at least 96% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence at least 97% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence at least 98% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence at least 99% identical to SEQ ID NO: 119. In some embodiments, the mRNA molecule comprises a sequence identical to SEQ ID NO: 119.C. Formulations and delivery

[0218] In some embodiments, the mRNA of the present invention is formulated to improve delivery. For example, and without limitation, the mRNA can be formulated in lipid nanoparticles (LNPs), polymer-based nanoparticles, lipid-polymer based nanoparticles virus like particles (VLPs), and engineered exosomes. In some embodiments, the mRNA is delivered without any carrier molecules, or naked. In some embodiments, the mRNA is delivered in complex with cationic peptides or polymers.1. Lipid Nanoparticles ( LNPs )

[0219] In some embodiments, an mRNA molecule of the present invention is formulated in lipid nanoparticles (LNPs). LNP components are selected based on the desired target, cargo (e.g., mRNA molecules), size, and / or other desired feature. LNP components include,for example, ionizable lipids, helper lipids, sterols, and / or PEG-lipids. The relative amounts, or molar ratios, of ionizable lipid, helper lipid, cholesterol, and PEG-lipid are optimized for a given target or administration route. In some embodiments, the LNPs do not contain a targeting ligand. In some embodiments, the LNPs contain a targeting ligand.

[0220] In general, LNPs are small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers.

[0221] The LNPs of the present invention can be prepared with any method commonly known in the art. In some embodiments, the LNPs comprise a storage buffer. In some embodiments, the storage buffer comprises saline. In some embodiments, the storage buffer does not comprise saline.

[0222] LNP sizes vary. In some embodiments, the LNPs for formulating the mRNA molecules of the present disclosure have an average hydrodynamic diameter of 10-1000 nm (i.e., 10-100 nm, 10-150 nm, 10-200 nm, 50-100 nm, 50-120 nm, 50-150 nm, 60-90 nm, 60- 120 nm, 80-100 nm, 80-120 nm, 100-200 nm, 100-500 nm, 200-800 nm, 100-1000 nm, or 500-1000 nm). In some embodiments, the average hydrodynamic diameter is at least 10 nm, at least 20 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, at least 120 nm, or at least 150 nm. In some embodiments, the average hydrodynamic diameter is less than 20 nm, less than 50 nm, less than 60 nm, less than 70 nm, less than 80 nm, less than 90 nm, less than 100 nm, less than 110 nm, less than 120 nm, or less than 150 nm. In some embodiments, the average hydrodynamic diameter is about 10 nm, about 20 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, or about 150 nm. i. Ionizable Lipids

[0223] In some embodiments, the LNP comprises at least one ionizable lipid. Ionizable lipids generally contain an amine-containing group on the head group. In some embodiments, the ionizable lipid comprises an ionizable cationic lipid. In some embodiments, the ionizable lipid comprises an ionizable anionic lipid.

[0224] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2021 / 141969 Al (Hamilton et al.), the entirety of which is incorporated by reference herein.In some embodiments, the ionizable lipid comprises a compound of Formula (I) of WO 2021 / 141969 Al (Hamilton et al.).(Formula (I))

[0225] In some embodiments, R1in Formula (I) comprises C9-C20 alkyl or C9-C20 alkenyl with 1-3 units of unsaturation. For example, in some embodiments R1comprises a C9-C20 alkenyl with 2 units of unsaturation, such as, without limitation, a C17 alkenyl with 2 units of unsaturation.

[0226] In some embodiments, X3, X5, and X6in Formula (I) are independently absent.

[0227] In some embodiments, X1is -O-. In some embodiments, X1is absent.-(CH)a- I

[0228] In some embodiments, X2is X7. In some embodiments, X2is -(CH2)a- or - CH(OH)-. In some embodiments, a is an integer between 0 and 6. In some embodiments, a is 0, 1, 2, 3, 4, 5, or 6. In some embodiments, a is 0 and X2is absent. In some embodiments, a is 1.

[0229] In some embodiments, X7is independently hydrogen or hydroxyl. In some embodiments, X7is hydroxyl. In some embodiments, X7is hydrogen.

[0230] In some embodiments, X4is a 6-membered heterocyclyl optionally substituted with 1 or 2 Ci-Ce alkyl groups. In some embodiments, the heterocyclyl comprises at least one nitrogen. For example, some embodiments, X4is piperidinyl. In some embodiments, X4is ethylpiperidinyl.

[0231] In some embodiments, A1and A2are independently C5-C12 alkyl or C5-C12 alkenyl with 1-3 units of unsaturation. In some embodiments, A1and A2are independently C5-C12 alkenyl with 1 unit of unsaturation. In some embodiments, A1is Cs alkenyl with 1 unit of unsaturation. In some embodiments, A2is Cs alkenyl with 1 unit of unsaturation.

[0232] In some embodiments, nl is an integer between 1 and 6. In some embodiments, nl is 1, 2, 3, 4, 5, or 6. In some embodiments, nl is 2.

[0233] In some embodiments, the ionizable lipid is a compound selected from Table Cl. Table Cl. Exemplary ionizable lipids of Formula (I).

[0234] In some embodiments, the ionizable lipid is a compound of Formula (I) comprising 3-((((l-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)-2-(((4-(((Z)-oct-5-en-l-yl)oxy)-4-(((Z)- oct-5-en-l-yl)oxy)butanoyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Compound 1).(Compound 1)

[0235] In some embodiments, the ionizable lipid comprises a compound disclosed in WO 2022 / 140252 Al (Patwardhan et al.), the entirety of which is incorporated by reference herein. In some embodiments, the ionizable lipid comprises a compound of Formula (III-a-i) of WO 2022 / 140252 Al (Patwardhan et al.), or its N-oxide:m-s(Formula (III-a-i))

[0236] In some embodiments, R1is hydrogen.

[0237] In some embodiments, L1is C2-C6 heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. For example, in some embodiments, L1is a C4 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2CH2-. In some embodiments, L1is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.

[0238] In some embodiments, each R is independently C6-C12 alkyl or C6-C12 alkenyl with 1-3 units of unsaturation.

[0239] In some embodiments, each L is independently C1-C5 alkylenyl.

[0240] In some embodiments, each L2is independently C4-C8 alkylenyl.

[0241] In some embodiments, the ionizable lipid is a compound selected from Table C2. Table C2. Exemplary ionizable lipids of Formula (III-a-i).

[0242] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((3-hydroxypropyl)azanediyl)bis(heptane-7, 1-diyl) bis(4,4-bis(((E)-oct-5-en- 1- yl)oxy)butanoate) (Compound 37).(Compound 37)

[0243] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((2-hydroxyethyl)azanediyl)bis(hexane-6, 1-diyl) bis(6,6-bis(hexyloxy)hexanoate)(Compound 49).(Compound 49)

[0244] In some embodiments, the ionizable lipid is a compound of Formula (III-a-i) comprising ((2-hydroxyethyl)azanediyl)bis(heptane-7, 1-diyl) bis(4,4-bis(((Z)-oct-5-en-l- yl)oxy)butanoate) (Compound 36).(Compound 36)

[0245] In some embodiments, the ionizable lipid comprises a compound of Formula (I’ ’-a iii) of WO 2022 / 140252 Al (Patwardhan et al.).(Formula (I”-a-iii))

[0246] In some embodiments, R1is hydrogen.

[0247] In some embodiments, E1is C2-C6 heteroalkylenyl comprising at least 1 heteroatom. In some embodiments, the heteroatom is oxygen. In some embodiments, E1is a C3 heteroalkylenyl comprising 1 oxygen atom, such as, for example and without limitation, - OCH2CH2-.

[0248] In some embodiments, each R is independently C6-C12 alkyl or C6-C12 alkenyl with 1-3 units of unsaturation.

[0249] In some embodiments, R” is C6-C12 alkyl.

[0250] In some embodiments, each L is independently C1-C5 alkylenyl.

[0251] In some embodiments, each L2is independently C4-C8 alkylenyl.

[0252] In some embodiments, the ionizable lipid is a compound selected from Table C3.Table C3. Exemplary ionizable lipids of Formula (I”-a-iii).

[0253] In some embodiments, the ionizable lipid is a compound of Formula (I”-a-iii) comprising nonyl 8-((6-((4,4-bis(octyloxy)butanoyl)oxy)hexyl)(2- hydroxyethyl)amino)octanoate (Compound 82).(Compound 82)

[0254] In some embodiments, the ionizable lipid is a compound of Formula (I”-a-iii) comprising nonyl 8-((2-hydroxyethyl)(6-((4-(((Z)-oct-5-en-l-yl)oxy)-4-(((Z)-oct-5-en-l- yl)oxy)butanoyl)oxy)hexyl)amino)octanoate (Compound 81).(Compound 81)

[0255] The ionizable lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, Cl 2-200 and DLin-KC2-DMA.

[0256] In some embodiments, the ionizable lipid constitutes at least 40, 45, or 50 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 40 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 45 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 50 mole percent of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes at least 47 mole percent of the total moles of components in theLNP. In some embodiments, the ionizable lipid constitutes 47.5 mole percent of the total moles of components in the LNP.

[0257] In some embodiments, the ionizable lipid constitutes 40 mol% to 50 mol% of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes 45 mol% to 50 mol% of the total moles of components in the LNP. In some embodiments, the ionizable lipid constitutes 47 mol% to 48 mol% of the total moles of components in the LNP. ii. Sterols

[0258] Sterols can aid in stability and promote membrane fusion of the LNPs. In some embodiments, the LNP comprises at least one sterol. In some embodiments, the sterol comprises cholesterol. In some embodiments, the sterol is unmodified cholesterol. In some embodiments, the sterol comprises a variant of cholesterol. In some embodiments, the LNP comprises a derivative of cholesterol. Cholesterol variants may be side-chain or ring oxidized from enzymes acting on unmodified cholesterol. In some embodiments, the cholesterol is oxidized on the beta-ring structure or on the hydrocarbon tail structure. Cholesterols include, for example and without limitation, 25 -hydroxy cholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27 -hydroxy cholesterol, 6-keto-5a-hydroxycholesterol, 7-ketocholesterol, 7 - hydroxycholesterol, 7a-hydroxycholesterol, 7 P-25 -dihydroxy cholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof.

[0259] In some embodiments, the sterol constitutes 35 mol% to 45 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40 mol% to 45 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40 mol% to 41 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes at least 40 mol% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 41% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.25% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.5% of the total moles of components in the LNP. In some embodiments, the sterol constitutes 40.75% of the total moles of components in the LNP. iii. PEG-Lipids

[0260] PEG-lipids can reduce LNP aggregation, shield the LNPs from non-specific endocytosis, and reduce opsonization by serum proteins and reticuloendothelial clearance. Insome embodiments, the LNP comprises at least one PEG or PEG-modified lipid. In some embodiments, the LNP comprises at least one PEG-modified lipids. In some embodiments, the LNP comprises PEG. Alternatively, the PEG-lipids may be referred to as PEGylated lipids or PEG-lipids. In some embodiments, the PEGylation is reversible with the PEG moiety gradually releasing into blood circulation. PEG-lipids include, for example and without limitation, PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG- modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. Additional examples of PEG-lipids include, without limitation, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG, or PEG-DSPE lipids. In some embodiments, the PEGylation comprises PEGlk, PEG2k, PEG5k, or PEGlOk. In some embodiments, the LNP comprises PEG-DMG. In some embodiments, the PEGylation comprises PEG2k. In some embodiments, the LNP comprises DMG-PEG2k.

[0261] In some embodiments, the PEG-lipid constitutes 0 mol% to 5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 3 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1 mol% to 2 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2 mol% to 3 mol% of the total moles of components in the LNP.

[0262] In some embodiments, the PEG-lipid constitutes 1 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.25 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 1.75 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.25 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.5 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 2.75 mol% of the total moles of components in the LNP. In some embodiments, the PEG-lipid constitutes 3 mol% of the total moles of components in the LNP.

[0263] In some embodiments, the PEG is replaced by a different polymeric compound such as, but not limited to, polyethenes, poly(l-lysine)(PLL), PEG grafted to PLL, cationiclipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly (alkylene imines), a poly amine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobutyl)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, poly anhydrides, poly hydroxy acids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly (orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycy anoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine- containing polymers, dextran polymers, dextran polymer derivatives or combinations thereof. iv. Helper Lipid

[0264] Helper lipids in LNPs may contribute to stability and delivery efficiency and / or mitigate any toxicity from the ionizable lipids. In some embodiments, the helper lipid is a phospholipid (also known as neutral phospholipid). In some embodiments, the phospholipid comprises a phospholipid moiety and at least one fatty acid moiety. In some embodiments, the phospholipid comprises one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers.

[0265] Exemplary phospholipid moieties include, but are not limited to, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, the fatty acid moiety includes, for example and without limitation, lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, a-linoleic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docasohexaenoic acid. Non-natural species or natural species with modification and substitutions, including branching, oxidation, cyclization, and alkynes, are included. For example, a phospholipid may be modified with an alkyne, which could allow for copper-catalyzed cycloaddition, or click-chemistry, with an azide to functionalize the lipid bilayer. In some embodiments, the lipid bilayer is functionalized. In some embodiments, the lipid bilayer is not functionalized.

[0266] In some embodiments, the helper lipid is a lipid having cone-shape geometry, e.g., dioleoylphosphatidylethanolamine (DOPE). In some embodiments, the helper lipid is a cylindrical- shaped lipid such as phosphatidylcholine.

[0267] Helper lipids include, for example and without limitation, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylethanolamine (DOPE), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), l,2-(cis-cis-9,12-octadecadienoyl)-sn-glycero-3- phosphatidylcholine (DUPC), l-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1,2- di-O-octadecyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OchemsPC), 1-hexadecanoyl-sn- glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, l,2-diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocoline, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3- phosphoglycerol (DOPG), l,2-Dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1- hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (POPE), 1,2- Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-Dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE), l,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), l-octadecanoyl-2-(9Z- octadecenoyl)-sn-glycero-3-phosphocholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE). In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid is DMPC. In some embodiments, the helper lipid is DOPE.

[0268] In some embodiments, the helper lipid is non-cationic lipid.

[0269] In some embodiments, the helper lipid constitutes 5 mol% to 15 mol% of the total moles of components in the LNP. In some embodiments, the helper lipid constitutes 5 mol% to 10 mol% of the total moles of components in the LNP. In some embodiments, the helper lipid constitutes 10 mol% to 15 mol% of the total moles of components in the LNP. In someembodiments, the helper lipid constitutes 10 mol% of the total moles of components in the LNP. v. Exemplary LNP Formulations

[0270] In some embodiments, the LNP formulation comprises about 40 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 15 mol% helper lipid, about 35 mol% to about 45 mol% sterol, and about 0 mol% to about 5 mol% PEG-lipid. In some embodiments, the LNP formulation comprises about 45 mol% to about 50 mol% ionizable lipid, about 5 mol% to about 15 mol% helper lipid, about 40 mol% to about 45 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid. In some embodiments, the LNP comprises about 45 mol% to about 50 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% to about 45 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid. In some embodiments, the LNP comprises about 47 mol% to about 48 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% to about 41 mol% sterol, and about 1 mol% to about 3 mol% PEG-lipid.

[0271] In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40 mol% sterol, and about 2.5 mol% PEG-lipid. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40.75 mol% sterol, and about 1.75 mol% PEG-lipid. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 41 mol% sterol, and about 1.5 mol% PEG-lipid. In some embodiments, the LNP comprises about 47.5 mol% ionizable lipid, about 10 mol% helper lipid, about 40.5 mol% sterol, and about 2 mol% PEG-lipid.

[0272] In some embodiments, the LNP comprises Compound 1 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 37 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 49 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 36 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 82 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 81 as the ionizable lipid, DSPC as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid.

[0273] In some embodiments, the LNP comprises Compound 1 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 37 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 49 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 36 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 82 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid. In some embodiments, the LNP comprises Compound 81 as the ionizable lipid, DOPE as the helper lipid, cholesterol as the sterol lipid, and DMG-PEG2k as the PEG-lipid.

[0274] In some embodiments, the LNP comprises a composition selected from Table D.Table D. Exemplary LNP compositions.2. Other Delivery Vehicles i. Engineered Exosomes

[0275] Exosomes are tiny vesicles smaller than 50 nm secreted by mature reticulocytes, which are associated with transferrin receptors and function in antigen presentation during theregulation of immune cells. In some embodiments, engineered exosomes act as cargo carriers and deliver small hydrophilic or lipophilic molecules, including some therapeutic drugs to cells, participating in the regulation of many major diseases. Exosomes can improve bioavailability of some drugs when taken orally, reducing the total dose required for administration, and minimizing side effects. In some embodiments, the mRNA molecules discussed herein are delivered using engineered exosomes. ii. Viral Like Particles (VLPs)

[0276] In some embodiments, mRNA molecules discussed herein are delivered using viral delivery particles. Viral particles include recombinant viruses and viral like particles (VLPs). As used herein, the term Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self-assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. VLPs can be produced from different viruses, such as adeno-associated viruses, retroviruses, lentiviruses and vesiculoviruses. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells. VLPs possess diverse applications in therapeutics, immunization, and diagnostics. VLPs have been synthesized in a wide range of ESs, including prokaryotic (bacteria) and eukaryotic (insect cells, mammalian cell lines, plant cells, or yeast). The functionality of VLPs can be increased through modifying their exterior or interior surface by displaying the heterologous epitopes of interest using different methods like peptide conjugation, genetic fusion, and chemical crosslinking.

[0277] In some embodiments, the VLP is derived from a Vesiculovirus. In some embodiments, the VLP is derived from VSV (Indiana vesiculovirus, formerly Vesicular stomatitis Indiana virus (VSIV or VSV). In some embodiments, the virus like particle comprises a mutated VSV-G protein. VSV-G protein is a single transmembrane glycoprotein (G) which plays a critical role during the initial steps of virus infection, it is responsible for virus attachment to specific receptor, LDL-R. In the cell, G protein triggers the fusion between the viral and endosomal membranes, which releases the viral genome in the cytosol for the subsequent steps of infection. In some embodiments, VSV-G protein is mutated to abolish its binding to LDL-R receptor. Lor example, a VSV-G envelope protein may be a mutated at one or more of any one of H8, K47, Y209, and / or R354. In some embodiments, aVLP may comprise a mutated VSV-G protein described in the PCT patent application Publication No. WO2019057974; the contents of which are incorporated herein by reference in their entireties. In some aspects, the VLP for delivery of mRNA molecules is a viral particle disclosed in the PCT Publication NOs. WO2020236263 and WO2023107886; the contents of each of which are incorporated herein by reference in their entireties.

[0278] In some embodiments, the virus like particle is pseudotyped. As a non-limiting example, the virus like particle is VSV-G-pseudotyped lentiviruses (VSV-G-LVs).

[0279] In some embodiments, the viral particle for delivering mRNA molecules is a retrovirus, a recombinant AAV, or an adenovirus.D. Compositions

[0280] In one aspect, the present disclosure provides pharmaceutical compositions comprising one or more mRNA molecules described herein. Pharmaceutical compositions may include mRNA molecules as described herein in combination with one or more pharmaceutically or physiologically acceptable carrier, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline or phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions which may be used in the pharmaceutical compositions of the disclosure include, for example, DMSO. Compositions can be formulated for any suitable administration, e.g., for intravenous administration.

[0281] Pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient (e.g., mRNA molecules) into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0282] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would beadministered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0283] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) mRNA molecules described herein.

[0284] A pharmaceutical composition of the present disclosure may be prepared, packaged, and / or sold in a formulation suitable for administration by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g., by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray, nasal spray, and / or aerosol, and / or through a portal vein catheter. In some embodiments, RNAs, and / or pharmaceutical compositions thereof, are administered by systemic intravenous injection. In specific embodiments, mRNA molecules and / or pharmaceutical compositions thereof may be administered intravenously and / or orally. In specific embodiments, mRNA molecules and / or pharmaceutical compositions thereof may be administered intramuscularly.

[0285] Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances. In some embodiments, pharmaceutical compositions of the present invention may optionally comprise one or more additional prophylactic compounds.

[0286] In some embodiments, the therapeutically active substance is an adjuvant. In some embodiments, the prophylactic compound is an adjuvant. In some embodiments, the pharmaceutical composition does not include an adjuvant.

[0287] The adjuvant may be used to enhance antibody response and can comprise any acceptable immuno stimulatory compound. For example, adjuvants include cytokines, toxins, or synthetic compositions. Adjuvants may be formulated as oil-in-water emulsions, water-in- oil emulsions, mineral salts, polynucleotides, or natural substances. Additional examples of adjuvants include, without limitation, Freund's adjuvant, oil such as MONTANIDE® ISA51, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, alpha-interferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts (such as aluminum hydroxide or other aluminum compound), MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A,monophosphoryl lipid A (MPL), lipopeptides (e.g., Pam3Cys), RIBI (which contains three components extracted from bacteria), MPL, trehalose dimycolate (TDM), MHC antigens, and cell wall skeleton (CWS) in a 2% squalene / Tween 80 emulsion.

[0288] In some embodiments, the adjuvant may be encoded by a second mRNA molecule. In some embodiments, the pharmaceutical composition comprises an mRNA molecule of the present invention encoding the VZV gE antigen and a second mRNA molecule encoding an adjuvant.

[0289] In some embodiments, a biologic response modifier (BRM) is co-administered to enhance immune responses. BRMs can upregulate T cell immunity and downregulate suppressor activity. Exemplary BRMs include, without limitation, Cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); low-dose Cyclophosphamide (CYP; 300 mg / m2) (Johnson / Mead, NJ), cytokines (such as y-interferon, IL-2, or IL-12), or genes encoding proteins involved in immune helper functions (such as B-7).

[0290] In accordance with some embodiments, a method of administering pharmaceutical compositions comprising an alternative nucleic acid encoding a VZV antigen to be delivered to a subject in need thereof is provided. In some embodiments, compositions are administered to humans. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

[0291] In some embodiments, the compositions and vaccines of the present invention may be formulated into preparations in solid, semi-solid, liquid, lyophilized, frozen, or gaseous forms. In some embodiments, the compositions and vaccines are formulated as a sterile suspension for intramuscular injection. In some embodiments, the compositions and vaccines are lyophilized and reconstituted prior to administration to a patient using a reconstitution liquid, for example, an adjuvant suspension component (liquid).E. Use and Applications

[0292] The mRNAs encoding one or more VZV antigen polypeptides, compositions and vaccines comprising mRNA encoding one or more VZV antigen polypeptides, can be used to induce an VZV-specific immune response, including any humoral and / or cellular immune response.

[0293] Suitably, the immune response comprises an antibody response to one or more of the herpesvirus polypeptides. The antibody titer produced by pharmaceutical compositions,also referred to herein as vaccines, of the invention may be a neutralizing antibody titer. Antibody titer is determined or measured by enzyme-linked immunosorbent assay (ELISA); and / or by microneutralization assay for example as described.

[0294] In some embodiments, the mRNAs encoding one or more VZV antigen polypeptides, compositions and vaccines comprising mRNA encoding one or more VZV antigen polypeptides are for prevention of herpes zoster (HZ) (also known as shingles) in a human patient.

[0295] In some embodiments, the patients are adults aged 50 years or older. For examples, the patients are adults aged 60 years or older, adults aged 70 years or older.

[0296] In other embodiments, the patients are adults aged 18 years and older who are or will be at increased risk of HZ due to immunodeficiency or immunosuppression caused by known disease or therapy. Immunocompromised patients may include patients having but not limited to Autologous Hematopoeitic Stem Cell Transplant (auHSCT) recipients, hematologic malignancies, renal transplant, solid malignant tumors, and HIV.

[0297] In some embodiments, the mRNAs encoding one or more VZV antigen polypeptides, compositions and vaccines comprising mRNA encoding one or more VZV antigen polypeptides are for prevention of chickenpox.

[0298] The vaccine may be administered to the patient by any routes of administration. In some embodiments, the vaccine may be administered to the patient by intramuscular, intradermal, or subcutaneous administration.

[0299] In some embodiments, the vaccine is administered by intradermal administration.

[0300] In some embodiments, the vaccine is administered by intramuscular administration.

[0301] In some embodiments, the vaccine is administered by subcutaneous administration.

[0302] In some embodiments, the vaccine is administered intramuscularly.

[0303] In some embodiments, the vaccine is administered in a single dose.

[0304] In some embodiments, the vaccine is administered intramuscularly in a single dose.

[0305] In some embodiments, the vaccine is administered in multiple doses. In some embodiments, the vaccine is administered in two separate doses. As non-limiting examples, the vaccine is administered according to the following schedules: a first dose at Month 0 followed by a second dose administered 2 to 6 months later. In some embodiments, for patients who are or will be immunodeficient or immunosuppressed and who would benefit from a shorter vaccination schedule: A first dose at Month 0 followed by a second dose administered 1 to 2 months later.

[0306] Accordingly, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 50% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 60% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 70% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 80% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 90% in subjects aged 50 years and older. In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ (shingles) by at least 95% in subjects aged 50 years and older.

[0307] In some embodiments, the compositions and vaccines significantly reduce the risk of developing HZ in patients aged 18 years and older who are or will be immunodeficient or immunosuppressed.

[0308] In some embodiments. The vaccine efficacy against HZ maintains the same the first year, the second year, the third year, the fourth year and later after vaccination.

[0309] In another aspect, the mRNAs encoding one or more VZV antigen polypeptides, compositions and vaccines comprising mRNA encoding one or more VZV antigen polypeptides may be used for the prevention of Postherpetic Neuralgia (PHN) in a patient which can be attributed to the effect of the vaccine on the prevention of HZ.

[0310] In some embodiments, the compositions and vaccines may be administered concomitantly with another vaccine.EXAMPLES

[0311] Various aspects of the invention are described in further detail in the following Examples. The following examples describe some of the exemplary modes of making and practicing the present invention. However, it should be understood that these examples are for illustrative purposes only and are not meant to limit the scope of the invention.Example 1. Design and Production of mRNA VZV Vaccine Constructs

[0312] This example describes the antigen design, mRNA construct optimization, and testing for the mRNA VZV vaccine constructs. The terms “mRNA VZV vaccine constructs” and “mRNA VZV gE constructs” are used interchangeably throughout the application andrepresent the mRNA described herein that encode a VZV gE polypeptide. The term “mRNA VZV vaccine” refers to a composition comprising an mRNA VZV vaccine construct.Antigen Design

[0313] Glycoprotein E (gE) is one of the viral binding proteins for varicella zoster virus (VZV), making gE a strong target for the neutralizing antibodies induced by vaccines. The gE protein comprises a signal domain, an extracellular domain, a transmembrane domain, and an intracellular domain. The intracellular domain is responsible for receptor trafficking, including targeting to the trans-Golgi network, endocytosis, and phosphorylation while the extracellular domain is the domain responsible for viral attachment. Therefore, when optimizing the design of the antigen, the gE protein was truncated and / or mutated at various points to retain the extracellular domain and eliminate the regulatory motifs from the intracellular domain. The truncation points are visualized in FIG. 1. When applicable, a mutation (Y569A) was included. Although not bound by any theory, the Y569A mutation was designed to disrupt trans-Golgi network targeting.Codon Optimization

[0314] The open reading frame (ORF) sequences that encode the gE antigen polypeptides were codon-optimized to increase expression of the VZV gE antigen and therefore increase immune response to VZV gE antigen. Codon optimization was performed using one or more of the following strategies: 1) optimization for human codon usage, 2) optimization to increase GC content in addition to optimal codon usage, and 3) optimization for secondary structure stability in addition to optimal codon usage.Example 2. mRNA translation and antigen protein expressionConstruct Expression

[0315] Expression of exemplary mRNA VZV gE constructs was assessed in K562 lymphoblast cells electroporated with the respective mRNA constructs encoding VZV gE. Expression was determined by measuring VZV fluorescence intensity by flow cytometry. Fold-over-control (FOC) values were calculated for each construct by dividing the geometric mean of the sample by the geometric mean of the negative controls. As shown in FIG. 2, expression of exemplary constructs (gE A562, gE A562 IgGK, gE A568, gE A568 IgGK, and gE A574 Y569A described in Table A) outperformed a reference construct across the tested time points. Further, the percent of cells expressing VZV gE was higher for the exemplary constructs than the reference construct. mRNA VZV gE constructs with high expression wereselected for further testing in a second trial with similar methodology. Results from the second trial can be found in FIG. 3.

[0316] Overall, exemplary mRNA VZV gE constructs, including codon-optimized constructs, of the present invention displayed higher gE expression than a reference construct.Example 3. mRNA VZV Vaccine Constructs Induced gE-Specific Immune Response in Mice

[0317] This example tested the immunogenicity and tolerability of exemplary mRNA VZV gE constructs in a mouse model.

[0318] The study was carried out over 42 days with mRNA doses administered intramuscularly on Day 0 and Day 21 to C57BL / 6J mice. Blood was collected on Days 1, 21, and 42, and spleens were collected on Day 42. Table 1 provides the in vivo study design for these mRNA VZV vaccine constructs. Group 1 contained the vehicle, phosphate-buffered saline (PBS). The doses for groups 2 and 3 were molar equivalents, and the dose for Shingrix (group 4) was one tenth of the human dose.Table 1. Study design for mRNA VZV vaccine constructsDay 42 was quantified by ELISA. The IgG titers, including titers for IgG subclasses, are shown in FIG. 4A-FIG. 4D, including Total IgG (FIG. 4A), IgGl (FIG. 4B), IgG2b (FIG. 4C), and IgGc (FIG. 4D). The exemplary mRNA constructs of the present invention had comparable or higher IgG titers to Shingrix, a commercial recombinant protein VZV vaccine. The ratio of IgG2c titer to IgGl titer for each group was calculated as shown in FIG. 5A- FIG. 5B at both Day 21 (FIG. 5A) and Day 42 (FIG. 5B). Higher IgG2c / IgGl ratios are indicative of Thl type cellular immune response.

[0320] A plaque reduction neutralization test (PRNT) was also performed to assess neutralizing antibodies in the serum samples from Day 21 and Day 42. Serum samples were diluted and mixed with a viral suspension to allow the antibodies to react with the virus. The samples were then poured over a confluent monolayer of host cells. The surface of the celllayer was further covered in a layer of agar or carboxymethyl cellulose to prevent the virus from spreading indiscriminately. The concentration of plaque forming units (PFUs), or regions of infected cells, was estimated after a few days. The PRNT50 titer is the concentration of serum required to reduce the number of plaques by 50% compared to the serum free virus and represents the effectiveness of the antibodies in serum. As shown in FIG. 6, the PRNT50 titers for exemplary mRNA VZV gE constructs were comparable to or higher than the those of the Shingrix vaccine.

[0321] Spleen samples from Day 42 were assessed for VZV gE-specific T cell responses. The data for CD4 and CD8 T cell responses are shown in FIG. 7A-FIG. 7E and FIG. 8A- FIG. 8D, respectively. The quantification of CD4 T cell responses includes the frequency of CD4 T cells with IFNy secretion (FIG. 7A), TNF secretion (FIG. 7B), IL-2 secretion (FIG. 7C), IL-4 secretion (FIG. 7D), and interleukin-5 (IL-5) secreting CD4 T cells (EIG. 7E). The quantification of CD8 T cell responses includes the frequency of CD8 T cells with ILNy secretion (FIG. 8A), TNF secretion (FIG. 8B), IL-2 secretion (FIG. 8C), and polyfunctional CD8 cells (FIG. 8D). The exemplary mRNA VZV gE constructs generated higher responses than Shingrix in both CD4 and CD8 T cells.

[0322] Finally, the tolerability of exemplary mRNA VZV gE constructs was assessed. The body weight (FIG. 9A) and variation in body weight (FIG. 9B) were tracked over the duration of the study as shown in FIG. 9A-FIG. 9B. Injection site reactions were also graded on Day 1 (FIG. 10A) and Day 22 (FIG. 10B), the days after each dose. Grades were assigned as described in Table 2.Table 2. Grading scale for injection site reactions.

[0323] The number of mice per group assigned each grade is summarized in FIG. 10A- FIG. 10B. The mice that were administered mRNA VZV gE constructs had no injection site reactions after either dose, while every mouse administered Shingrix displayed at least a Grade 1 reaction after the second dose.

[0324] Overall, the exemplary mRNA VZV gE constructs resulted in higher Thl cellular immune responses than the recombinant protein vaccine, Shingrix, and showed better tolerability than Shingrix throughout the study.Example 4. mRNA VZV Vaccine Formulated in LNPs Induced gE-Specific Immune Response in Mice

[0325] This example demonstrates the immunogenicity and tolerability of exemplary mRNA VZV gE constructs formulated in LNPs.

[0326] The study was carried out over 35 days with mRNA VZV vaccine doses administered intramuscularly on Day 0 and Day 21 to C57BL / 6 mice. Blood was collected on Days 1, 21, and 35, and spleens were collected on Day 35. Table 3 provides the in vivo study design for these mRNA VZV vaccine constructs formulated in LNPs. The study was completed at an mRNA dose of 10 pg and repeated at an mRNA dose of 1 pg. The Vehicle was Tris Sucrose Saline (TSS) buffer.Table 3. Study design for mRNA VZV vaccine formulated in LNPs

[0327] ELISA assays were performed on the samples from Day 21 and Day 35. The resulting IgG titers for each of the Groups 1-10 are shown in FIG. 11A-FIG. 11B and FIG. 12A-FIG. 12B for the high dose (Day 21 FIG. 11A, Day 35 FIG. 11B) and low dose (Day 21 FIG. 12A, Day 35 FIG. 12B) groups, respectively. As shown, LNPs containing Compound 49 resulted in higher IgG titers across both dose groups.

[0328] Spleen samples from Day 35 were assessed for VZV gE-specific T cell responses. Results for the CD4 and CD8 T cell responses are shown in FIG. 13A-FIG. 13C and FIG.14A-FIG. 14C, respectively. The quantification of CD4 T cell responses includes the frequency of CD4 T cells with IFNy secretion (FIG. 13A), TNF secretion (FIG. 13B), and IL-2 secretion (FIG. 13C). The quantification of CD8 T cell responses includes the frequency of CD8 T cells with IFNy secretion (FIG. 14A), TNF secretion (FIG. 14B), and IL-2 secretion (FIG. 14C). Exemplary LNPs containing Compound 1 induced greater responses for both CD4 and CD8 T cells as compared to Shingrix.

[0329] Finally, the tolerability of exemplary LNPs containing mRNA VZV gE constructs was assessed. The body weight and variation in body weight were tracked over the duration of the study as shown in FIG. 15A-FIG. 15C, following administration of either high dose (FIG. 15A) or low dose (FIG. 15B). Injection site reactions were also graded on Day 1 and Day 22, the days after each dose. Grades were assigned as described in Example 3. The number of mice per group assigned each grade is summarized in FIG. 16 for the high dose group (Day 1 FIG. 16A, Day 22 FIG. 16B) and FIG. 17 for the low dose group (Day 1 FIG. 17A, Day 22 FIG. 17B). At both dose levels, mRNA VZV gE constructs formulated in LNPs show better tolerability than Shingrix.

[0330] Overall, this example demonstrated that exemplary mRNA VZV gE constructs formulated in LNPs induced VZV gE-specific T cell responses with good tolerability.Example 5. mRNA VZV Vaccine Administered In Vivo was Safe and Efficacious

[0331] This example demonstrates the safety and efficacy of exemplary mRNA VZV vaccines with single or double dosing. The exemplary mRNA VZV vaccines were also tested in a mouse model that were primed with a live, attenuated varicella vaccine to model a population that already had chickenpox. The exemplary mRNA VZV vaccines were formulated in LNP comprising Compound 1 as the ionizable lipid.

[0332] Vaccine dosing was studied in vivo in C57BF6J mice. The study design is described in Table 4 below and the study was completed over 123 days. Groups 1-4, 9-10, and 13 were primed with 250 pl (675 plaque-forming units (PFU)) of Varivax®, a live, attenuated varicella vaccine to model a population that already had chickenpox (“Chickenpox Model”). Groups 5-8, 11-12, and 14 were not primed with Varivax®. In the Chickenpox Model groups, attenuated varicella was administered on Day 0, and the first boost (1° boost) of either an exemplary mRNA VZV vaccine or recombinant protein VZV vaccine (Shingrix®) was administered on Day 28, and the second boost (2° boost) was administered on Day 49. All doses were administered intramuscularly, and each testing group had a samplesize of 8. Results are described for post-dosing with single dose (PD1) D21 samples and postdosing with two doses (PD2) D35 samples in the various groups tested.Table 4. Study design for exemplary mRNA VZV vaccine dosing

[0333] Blood samples were collected on Day 0, Day 28, Day 49, Day 53, Day 67, Day 81, Day 95, and Day 123 / 124 timepoints and analyzed for binding and neutralizing antibodies by ELISA and PRNT. Spleen samples were collected at the end of the study. At the terminal timepoint (Day 123 / 124), cytokine production following ex vivo stimulation of splenocytes with VZV gE peptide were detected by FACS analysis. Spleen samples were assessed by T- cell flow panels for VZV- specific T cell responses.

[0334] In an interim read-out, blood was collected on Day 53 after the 2° boost and analyzed for total IgG as shown in FIG. 18A and FIG. 18B. Total IgG was increased in groups that were primed with the attenuated varicella. The 2° boost also increased total IgG as compared to the testing groups without the 2° boost.

[0335] By the end of the study, all testing groups elicited strong humoral responses, as measured by anti- VZV gE antibody on Days 49, 53, 67, 81, 95, and 124 when compared to Group 13 (Varivax®) and Group 14 (vehicle). FIG 41A-FIG. 41Dshows the total IgG anti-VZV gE in serum from tested timepoints. From the Day 67 timepoint until the end of the study, higher responses were observed after a second dose administration in Groups 2, 6, 8, and 10 compared to their respective single dose counterparts in Groups 1, 5, 7, and 9. From Day 49 through the end of the study, administration of I O g mRNA7 given once or twice, either in combination with Varivax® (Group 3 and 4), or alone (Groups 7 and 8) generally resulted in higher responses in total IgG, IgG2b and IGg2c, when compared to respective counterpart groups in which mRNA7 was given either once or twice at I pg (Groups 1, 2, 5 and 6, respectively). In summary, a two-dose regimen of either high dose mRNA7 (10 pg) or Shingrix® (5 ug) generated comparable antibody titers.

[0336] Similar to the anti- VZV gE antibody assessment, Groups 1 to 12 elicited higher neutralization antibody titers response compared to Group 13 (Varivax®) and Group 14 (vehicle) measured on Days 49, 53, 67, 81, 95 and 124. All groups elicited strong humoral responses measured on Days 49, 53, 67, 81, 95 and 124 when compared to Group 13 (Varivax®) and Group 14 (vehicle). FIG. 42A-FIG. 42D shows the VZV neutralizing antibody in serum from tested timepoints. Higher titers responses were observed following a second dose administration in Groups 2, 4, 6, 8, and 10 when compared with respective single dose counterpart Groups 1, 3, 5, 7, and 9.

[0337] Ex vivo stimulation with VZV gE peptide showed a prominent Thl cytokine response on both the CD4+ T cells and CD8+ T cells. Overall, expression of CD4 Thl- mediated cytokine response was significantly high in the RNA vaccine groups Varivax® / 10pg mRNA7 (Group 4) and lOpg mRNA7 (Group 8).

[0338] One aim of the study was to evaluate whether mice primed with Varivax® generated elevated immune responses after a single or two-dose administration of the mRNA7 vaccine compared with Shingrix®. This study showed that the Varimax® priming had no impact on antibody titers after either a single or two-dose administration of mRNA7 VZV vaccine or Shingrix® compared to Vehicle.

[0339] In this study, no major effects of the mRNA VZV vaccines were observed on body weight and clinical signs. Body weight and clinical signs were monitored on the day of immunization and the day after or subsequently approximately every other day throughout the study. Clinical signs that were monitored included decreased activity, hunched posture, dehydration, erected fur, shallow breathing, eyes partly closed, swollen hind limbs, amongst other signs. Body weight measurements and body weight variation showed that there was a slight weight loss after immunizations on Days 0, 28 and Day 49, but all animals gainedweight thereafter, and no significant differences were observed between groups. In conclusion, no adverse effects were noted for the mRNA VZV vaccines in this in vivo studyExample 6.VZV mRNA preparation and purification

[0340] The DNA template that contains a DNA-dependent RNA polymerase promoter and the corresponding sequence for the mRNA construct were amplified and the extracted DNA samples were linearized and used as templates for in vitro transcription (IVT) to produce mRNA. In vitro transcript mixtures were then treated with DNase to degrade the DNA template. The resulting small DNA fragments can then be easily separated from larger mRNA molecules by tangential flow filtration (TFF) or molecular weight-cutoff spin filtration during purification. Following the DNase reaction, mRNA was purified from the impurities and materials used in the previous steps including endotoxins, immunogenic double stranded RNA (dsRNA), residual DNA template, RNA polymerase, excess NTPs, and elemental impurities. The treated samples were diluted in dT buffer and run by Oligo-dT Liquid Chromatography (LC) (Oligo(dT) affinity chromatography). The oligo(dT) specifically hybridizes to the polyA tails of mRNA. The contaminants were removed during the washing steps. Next, the immobilized mRNAs were released from the column using an elution buffer. The collected eluates were prepared again using RP buffer and run by RP- HPLC-FT (reversed-phase high performance liquid chromatography) for further purification. The collected samples were diluted in PBS. Tangential flow filtration (TFF) was applied to further purify the mRNA samples. In certain cases, IVT samples after DNase treatment were directly purified by Oligo-dT Liquid Chromatography (LC) (Oligo(dT) affinity chromatography), followed by molecular weight-cutoff spin filtration.

[0341] Alternatively, in vitro transcription mixtures were first treated with DNase to degrade the DNA templates. The treated samples were precipitated using a LiCI containing solution. The LiCI precipitants (mRNA pellets) were resuspended and run through RP- HPLC. The mRNA collections were further purified using TFF.5’ Capping

[0342] Exemplary mRNA VZV constructs were prepared with different 5’ caps and analyzed for the effect of 5’ capping on immunogenicity. The caps were added using CleanCap® methodology, a one-pot co-transcriptional capping method. Any method or reagent known in the art may be equivalently used for generating the capped mRNA. The preparation process was further optimized by reducing the concentration of CleanCap® reagent without significantly impairing capping efficiency. The study design, with differentexemplary 5’ caps and buffer concentrations are shown in Table 5. The study was then repeated with an mRNA dose of 1 pg.Table 5. Study design for exemplary 5’ Caps and production processes

[0343] For both the 10 pg and 1 pg dose levels, the alternative 5’ caps, G-AG 3’ OMe and G-m6AG, resulted in higher IgG titers than the G-AG caps as shown in FIG. 19A- FIG. 19B and FIG. 20A-FIG. 20B. The IgG2c / IgGl ratios were calculated as shown in FIG. 21A- FIG. 21B and FIG. 22A-FIG. 22B. At the lower dose, the alternative 5’ caps had no significant effect on the IgG2c / IgGl ratio. At both dose levels, the concentration of CleanCap® can be reduced without significant impact on IgG titers as shown in FIG. 23A- FIG. 23B and FIG. 24A-FIG. 24B.

[0344] Overall, this example demonstrated the efficacy of exemplary 5’ caps in increasing the IgG titers from administration of the mRNA VZV vaccines.Example 7. LNP Formulation for mRNA VZV Vaccines

[0345] This Example describes the optimization of exemplary LNP formulations for mRNA VZV vaccines. Additional LNP formulations can be found in WO 2021 / 141969 Al (Hamilton et al.) and WO 2022140252 Al (Patwardhan et al.), the disclosure of each of which is hereby incorporated by reference in its entirety.Exemplary LNP Formulations

[0346] Exemplary LNPs containing Compound 1, Compound 49, and Compound 82 as the ionizable lipid were prepared with different component ratios as described below in Table 6.Table 6. Exemplary LNP formulations for mRNA VZV vaccinesBuffer Screening

[0347] Buffer compositions for the LNPs were screened and evaluated based on (1) osmolality, (2) stability, and (3) anti-VZV gE response. Exemplary buffers comprising trisbuffered saline (TBS), 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES), HEPES saline, 3-(N-morpholino)propanesulfonic acid (MOPS), Tris, N- [Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES), and phosphate-buffered saline (PBS) were prepared as outlines in Table 7. The osmolality for each buffer was also measured. In this study, the target osmolality range for an injectable product was 300-500mOsm / kg with a firm upper limit of 600 mOsm / kg in order to avoid pain and irritation at the injection site.Table 7. Exemplary buffer compositions and osmolality with Compound 36 LNPsStability

[0348] The stability of the LNPs in each buffer was assessed by characterizing LNP size, polydispersity index (PDI), and encapsulation efficiency (EE%) after freeze thaw (FT) cycles and long-term storage. First, stability was compared after 0, 1, 3, or 5 FT cycles. As shown in FIG. 25, increasing buffer concentration to 100 mM generally resulted in an increase in ENP size after FT cycles. However, the 20 mM HEPES, MOPS, and TES buffers maintained the EE% better than their 100 mM counterparts. Then, the percent change in LNP size, PDI, and EE% was calculated for the exemplary buffer compositions, comparing the stability at aconstant 4°C to that after one FT cycle at -80°C. As shown in FIG. 26, the 100 mM HEPES Saline buffer had the highest increase in size after the FT cycle. Sucrose concentrations did not appear to affect stability.

[0349] The long-term stability of LNPs in exemplary buffer concentrations was measured after 0, 7, or 30 days of storage at 4°C. Most of the storage buffers maintained LNP stability after 7 days, as shown in FIG. 27. Notably, 20 mM HEPES best maintained the EE% after 30 days of storage.

[0350] Select buffers were further tested to assess stability after a FT cycle with LNPs of a different composition. The tested buffers maintained Compound 49 LNP stability as well as seen in FIG. 28.

[0351] Next, the impact of the acidification buffer and nitrogemphosphate ratio (N / P) was assessed for Compound 49 and Compound 1 LNP formulations. As shown in FIG. 29, the acetate buffer decreased the LNP size without affecting EE% for Compound 1 in LNP formulations. However, for Compound 49 LNP formulations, the acetate buffer decreased EE% while increasing LNP size as seen in FIG. 30. Additionally, for Compound 1 LNP formulations, increasing the pH of the acidifying buffer or N / P did not affect the LNP size, and for Compound 49 LNP formulations, increasing the pH of the acidifying buffer decreased the LNP size without affecting the EE%.

[0352] Finally, buffer compositions with LNP mRNA VZV vaccines were tested in vitro by delivering the LNP formulations to K562 lymphoblast cells. As shown in FIG. 31A and further quantified with area under the curve (AUC) in FIG. 31B, all tested buffers had comparable population percents of VZV gE positive cells. However, the buffers did show variation in geometric mean fluorescence intensity (gMFI) as seen in FIG. 32A, further exemplified with AUC analysis shown in FIG. 32B.Example 8. In vivo Buffer Screen for Exemplary mRNA VZV Vaccine LNP formulations

[0353] In this study, exemplary LNP formulations in various buffers were tested in vivo in C57BI / 6J mice. Acidification buffers were modified and screened to optimize LNP characteristics and stability while ensuring the immune response was not impacted. Saline was also included in some exemplary buffers to improve potency for local administration.

[0354] The study was carried out over 35 days, and vaccine doses were administered intramuscularly on Day 0 and Day 21. Serum samples were taken on Day 0 (hour 0 and hour 6), Day 1, Day 20, Day 21 (hour 6), Day 22, and Day 35. Spleen samples were collected onDay 35. Testing groups and dosing are described in Table 8. Each group had a sample size of5.Table 8. Study design for mRNA VZV vaccine LNP formulations and buffer screening

[0355] Serum samples were analyzed for VZV gE- specific IgG titers by ELIS As andLuminex assays. Spleen samples were assessed by T-cell Flow Panels to determine VZV gE- specific T-cell responses.

[0356] First, the tolerability of exemplary LNP formulations was assessed. The body weight and variation in body weight were tracked over the duration of the study as shown in FIG. 33A-FIG. 33B. Injection site reactions were also graded on Day 1 and Day 22, the daysafter each dose. Grades were assigned as described in Example 3. The number of mice per group assigned each grade is summarized in FIG. 34A-FIG. 34B at both Day 1 (FIG. 34A) and Day 22 (FIG. 34B).

[0357] ELISA assays were performed on the samples from Day 21 and Day 35. The resulting total IgG titers are displayed in FIG. 35A-FIG. 35B. The IgGl, IgG2b, and IgG2c titers from Day 35 are shown in FIG. 36A-FIG. 36B. Exemplary LNP formulation LNP029, with DOPE as the helper lipid, resulted in high IgG titers on Day 35. Additionally, exemplary LNP formulations with saline in the storage buffer surprisingly showed increased IgG titers on Day 35. Increasing the pH of the acidification buffer caused the LNP particle size to decrease, however, the smaller particles still maintained potency.

[0358] The IgG2c / IgGl ratios for each group are shown in FIG. 37. The IgG2c / IgGl ratio is representative of the type of immune response induced by the exemplary mRNA VZV vaccine. A higher ratio indicates a Thl type cellular immune response, whereas a lower ratio is indicative of a Th2 type humoral immune response. The balance of the Thl and Th2 immune responses may improve the protective effect of the vaccine. Exemplary mRNA VZV vaccine formulations with LNP007 generally have a higher Thl type cellular immune response on Day 35.

[0359] Finally, spleen samples from Day 35 were assessed for VZV gE-specific T cell responses. Exemplary LNP formulations LNP007 and LNP029 lead to higher concentrations of IFNy -secreting CD4+ T cells, as shown by FIG. 38A, and TNFa -secreting CD4+ T cells, as shown by FIG. 38C. However, higher concentrations of IL-2 were not observed (FIG. 38B). Exemplary LNP formulations LNP007 and LNP029 also resulted in higher concentrations of IFNy -secreting and TNFa -secreting CD8+ T cells, as shown by FIG. 39A and FIG. 39C, but not higher concentrations of IL-2 (FIG. 39B).Example 9. Exemplary LNP Formulations of mRNA VZV Vaccines Demonstrated Safe and Efficacious Pharmacokinetic Profiles

[0360] This Example describes an in vivo study to assess the lipid pharmacokinetic profiles of lipid nanoparticles containing ionizable lipids Compound 1 and Compound 49. Exemplary LNP formulations, LNP007 and LNP011, were administered to mice via intramuscular injection at an mRNA dose of 10 pg.

[0361] The amount of lipid remaining in serum, liver, and injection site muscle was assessed using mass spectrometry at various timepoints post-injection. As shown in FIG. 40A-FIG. 40C, the data showed that intramuscular administration of exemplary mRNA VZVvaccines formulated in LNPs containing Compound 1 and Compound 49 ionizable lipids displayed pharmacokinetic profiles that allowed for safe and efficacious administration. Both LNPs containing Compound 1 and LNPs containing Compound 49 were below or the limit of detection (that is, the lowest limit of quantitation (LLoQ)), in the serum and liver by 7 days (168 hours) post- administration. LNP007 and LNP011 demonstrated similar clearance in serum (FIG. 40A), while LNP007 demonstrated more rapid clearance in the liver (FIG. 40B) and injection site muscle (FIG. 40C).

[0362] Taken together with Example 8, these results supported exemplary mRNA formulated in LNP formulations containing Compound 1 and Compound 49 for use as a vaccine for treating shingles.The present disclosure comprises sequences disclosed in Table 9, and variants thereof. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 90%, 95%, 96%, 97%, 98%, 99% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 90% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 95% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 96% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 97% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 98% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises at least 99% identity to any one of the sequences in Table 9. In some embodiments, the full length mRNA VZV vaccine sequence comprises 100% identity to any one of the sequences in Table 9.Table 9. Exemplary mRNA VZV vaccine full length sequencesEQUIVALENTS AND SCOPE

[0363] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

Claims

CLAIMSWhat is claimed is:

1. A messenger ribonucleic acid (mRNA) comprising a codon-optimized open reading frame (ORF) sequence that encodes a varicella- zoster virus (VZV) glycoprotein E (gE) polypeptide, wherein the codon-optimized ORF sequence is at least 90% identical to any one of the sequences of SEQ ID NO: 1-95.

2. The mRNA of claim 1, wherein the codon-optimized ORF sequence is at least 95% identical to any one of the sequences of SEQ ID NO: 1-95.

3. The mRNA of claim 2, wherein the codon-optimized ORF sequence comprises any one of the sequences of SEQ ID NO: 1-95.

4. The mRNA of claim 3, wherein the codon-optimized ORF sequence comprises SEQ ID NO: 21.

5. The mRNA of claim 3, wherein the codon-optimized ORF sequence comprises SEQ ID NO: 24.

6. The mRNA of any one of claims 1-5, wherein the mRNA further comprises a 5’ untranslated region (5’ UTR), a 3’ UTR, and / or a poly-adenine (poly- A) sequence.

7. The mRNA of claim 6, wherein the mRNA comprises, from the 5’ end to 3’ end, a) the 5’ UTR, b) the ORF sequence, c) the 3’ UTR, and d) the poly(A) sequence.

8. The mRNA of any one of claims 1-7, wherein the mRNA further comprises a 5’ cap.

9. The mRNA of claim 8, wherein the 5’ cap is a modified CAP1 comprising (7’methyl-3’-O- methyl)Guanosine-ppp-(N-6 methyl- 2’-O-methyl)Adenosine-Guanosine ((m7-3OMe)G-ppp- (m6-2OMe)AG).

10. The mRNA of claim 8, wherein the 5’ cap is a modified CAP1 comprising (7’methyl-3’- O-methyl)Guanosine-ppp- (2’-O-methyl)Adenosine-Guanosine ((m7-3OMe)G-ppp-AmG).

11. The mRNA of any one of the preceding claims, wherein the mRNA comprises a sequence that is at least 90%, or at least 95%, or 100% identical to any one of SEQ ID NO: 96-190.

12. The mRNA of claim 11, wherein the mRNA comprises the sequence of SEQ ID NO: 116.

13. The mRNA of claim 11, wherein the mRNA comprises the sequence of SEQ ID NO: 119.

14. The mRNA of any one of the preceding claims, wherein the codon-optimized ORF encodes a VZV gE polypeptide variant.

15. The mRNA of claim 14, wherein the VZV gE polypeptide variant is a truncated gE polypeptide lacking a carboxy terminal domain.

16. The mRNA of any one of the preceding claims, wherein the VZV gE polypeptide comprises a human IgG kappa (IgGK) signal peptide.

17. The mRNA of any one of the preceding claims, wherein the mRNA is chemically unmodified.

18. The mRNA of any one of claims 1-16, wherein the mRNA comprises at least one chemical modification.

19. The mRNA of claim 18, wherein the uracil nucleosides of the mRNA molecule are chemically modified.

20. The mRNA of claim 19, wherein the uracil nucleosides of the mRNA molecule are at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% chemically modified.

21. The mRNA of any one of claims 18-20, wherein the chemical modification is in the sugar subunit of the nucleoside.

22. The mRNA of any one of claims 7-21, wherein the poly(A) sequence is further modified with 1-20 ribonucleic acids that are not adenosine.

23. The mRNA of claim 22, wherein the poly (A) sequence is modified by 1, 2, 3, 4, or 5 guanosines.

24. The mRNA of any one of the preceding claims, wherein the mRNA is formulated in lipid nanoparticles (LNPs).

25. The mRNA of claim 24, wherein the LNP is stored in a storage buffer comprising saline.

26. The mRNA of claim 24 or claim 25, wherein the LNP comprises an ionizable lipid, a helper lipid, cholesterol, and a polyethylene glycol (PEG) -modified lipid, wherein the ionizable lipid comprises a compound in Table Cl, C2, or C3.

27. The mRNA of claim 26, wherein the ionizable lipid is:(Compound 49).

28. The mRNA of any one of claims 24-27, wherein the LNP comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41% cholesterol, and 1.5-2.5 % PEG-modified lipid.

29. The mRNA of claim 28, wherein the LNP comprises a molar ratio of: a) 47.5 % ionizable lipid, 10 % helper lipid, 40% cholesterol, and 2.5 % PEG- modified lipid, b) 47.5 % ionizable lipid, 10 % helper lipid, 40.25% cholesterol, and 2.25 % PEG- modified lipid,c) 47.5 % ionizable lipid, 10 % helper lipid, 40.5% cholesterol, and 2 % PEG- modified lipid, d) 47.5 % ionizable lipid, 10 % helper lipid, 40.75% cholesterol, and 1.75 % PEG- modified lipid, or e) 47.5 % ionizable lipid, 10 % helper lipid, 41% cholesterol, and 1.5% PEG-modified lipid.

30. The mRNA of claim 28 or claim 29, wherein the helper lipid is 1,2-distearoyl-sn-glycero- 3 -phosphocholine (DSPC), and wherein the PEG-modified lipid is 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

31. The mRNA of claim 28 or claim 29, wherein the helper lipid is 1,2-Dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), and wherein the PEG-modified lipid is DMG-PEG2K.

32. The mRNA of any one of claims 25-31, wherein the LNP comprises: i) a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or ii) a molar ratio of 47.5 % Compound 49, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or iii) a molar ratio of 47.5 % Compound 1, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or iv) a molar ratio of 47.5 % Compound 49, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K; and wherein Compound 1 is:; and wherein Compound 49 is:

33. A pharmaceutical composition comprising at least one of the mRNA of any one of the preceding claims and a pharmaceutically acceptable carrier.

34. A vaccine comprising the mRNA of any one of claims 1-32, or the pharmaceutical composition of claim 33.

35. The vaccine of claim 34, further comprising an adjuvant.

36. A vector for making the mRNA of any one of claims 1-34.

37. The vector of claim 36, wherein the vector is a non- viral DNA vector.

38. The vector of claim 36, wherein the vector is a viral vector.

39. The vector of any one of claims 36-38, further comprising a promoter sequence.

40. A composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella-zoster virus (VZV) glycoprotein (gE) polypeptide formulated in a lipid nanoparticle in an effective amount to induce in a human subject an immune response to the VZV gE polypeptide, wherein the lipid nanoparticle comprises a molar ratio of 47.5 % ionizable lipid, 10 % helper lipid, 40-41 % cholesterol, and 1.5-2.5 % PEG-modified lipid.

41. The composition of claim 40, wherein the ionizable lipid is:(Compound 1); or(Compound 49).

42. A composition comprising a messenger ribonucleic acid (mRNA) comprising an open reading frame (ORF) sequence encoding a varicella-zoster virus (VZV) glycoprotein (gE) polypeptide, wherein the mRNA is formulated in a lipid nanoparticle comprising an ionizable lipid, and wherein the ionizable lipid is:(Compound 49).

43. The composition of claim 42, wherein the LNP further comprises a helper lipid, cholesterol and a PEG-modified lipid.

44. The composition of any one of claims 40-43, wherein the helper lipid is DSPC, and wherein the PEG-modified lipid is DMG-PEG2K.

45. The composition of claim 44, wherein the LNP comprises, i). a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 %DMG-PEG2K; or ii) a molar ratio of 47.5 % Compound 49, 10 % DSPC, 40.75% cholesterol, and 1.75 %DMG-PEG2K; or iii) a molar ratio of 47.5 % Compound 1, 10 % DOPE, 40.75% cholesterol, and 1.75 %DMG-PEG2K; or iv) a molar ratio of 47.5 % Compound 49, 10 % DOPE, 40.75% cholesterol, and 1.75 %DMG-PEG2K; and wherein Compound 1 is:wherein Compound 49 is:

46. The composition of any one of claims 40-45, wherein the mRNA further comprises a 5’ cap, a 5’ untranslated region (5’ UTR), a 3’ untranslated region (3’ UTR), and / or a poly(A) sequence.

47. The composition of claim 46, wherein the mRNA comprises, from the 5’ end to 3’ end, a) the 5 ’UTR; b) the ORF sequence; c) the 3 ’UTR; and d) the poly(A) sequence.

48. The composition of any one of claims 40 - 47, wherein the ORF encodes a VZV gE polypeptide variant.

49. The composition of claim 48, wherein the VZV gE polypeptide variant is a truncated gE polypeptide lacking the carboxy terminal domain.

50. The composition of any one of claims 40-49, wherein the VZV gE polypeptide comprises a human IgG kappa signal peptide.

51. The composition of any one of claims 40-50, wherein the ORF sequence is codon- optimized.

52. The composition of claim 51, wherein the ORF sequence comprises a sequence that is at least 90%, or at least 95%, or 100% identical to any one of SEQ ID NO.: 1-95.

53. The composition of claim 52, wherein the mRNA comprises a sequence that is at least 90%, or at least 95%, or 100% identical to any one of SEQ ID NO: 96-190.

54. The composition of claim 52, wherein the mRNA comprises a sequence that at least 90%, or at least 95%, or 100% identical to any one of SEQ ID NO: 11655. The composition of claim 52, wherein the mRNA comprises a sequence that at least 90%, or at least 95%, or 100% identical to any one of SEQ ID NO: 119.

56. A kit comprising at least one of the mRNA of any one of claims 1-34, or the composition of any one of claims 40-55.

57. A vaccine comprising the composition of any one of claims 40-55.

58. The vaccine of claim 57, wherein the vaccine is a prophylactic vaccine.

59. A method for stimulating an immune response to varicella- zoster virus (VZV) in a human subject comprising administering to the human subject an effective amount of a messenger ribonucleic acid (mRNA) comprising a codon-optimized open reading frame sequence encoding a varicella- zoster virus (VZV) glycoprotein (gE) polypeptide, wherein the codon- optimized ORF sequence is at least 90%, or at least 95%, or 100% identical to any one of the sequences of SEQ ID NO: 1-95.

60. The method of claim 59, wherein the codon-optimized ORF sequence comprises SEQ ID NO: 21.

61. The method of claim 59, wherein the codon-optimized ORF sequence comprises SEQ ID NO: 24.

62. The method of any one of claims 59-61, wherein the administration is for prophylactic treatment.

63. The method of any one of claims 59-61, wherein the immune response is a neutralizing antibody response to the glycoprotein (gE) polypeptide encoded by the mRNA, and / or a cellular immune response to the glycoprotein (gE) polypeptide encoded by the mRNA.

64. The method of any one of claims 59-63, wherein the mRNA is formulated in a lipid nanoparticle.

65. The method of claim 64, wherein the lipid nanoparticle comprises an ionizable lipid, a helper lipid, cholesterol, and a PEG-modified lipid, wherein the ionizable lipid is:(Compound 49).

66. The method of claim 65, wherein the helper lipid is DSPC, and wherein the PEG- modified lipid is DMG-PEK2K.

67. The method of claim 66, wherein the mRNA is formulated in the LNP comprising: i). a molar ratio of 47.5 % Compound 1, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or ii) a molar ratio of 47.5 % Compound 49, 10 % DSPC, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or iii) a molar ratio of 47.5 % Compound 1, 10 % DOPE, 40.75% cholesterol, and 1.75 % DMG-PEG2K; or iv) a molar ratio of 47.5 % Compound 49, 10 % DOPE, 40.75% cholesterol, and 1.75 %DMG-PEG2K; and wherein Compound 1 is:wherein Compound 49 is:

68. The method of any one of claims 59-67, wherein the mRNA is administered with a single dose.

69. The method of any one of claims 59-68, wherein the mRNA is administered to the human subject via intramuscular administration, intradermal administration, or subcutaneous administration.

70. A method for treating or preventing a disease caused by varicella- zoster virus (VZV) comprising administering to a human subject in need thereof the mRNA of any one of claims 1-32, or the vaccine of any one of claims 34-35 and 57-58, or the composition of any one of claims 40-55.

71. The method of claim 70, wherein the administration is to prevent primary VZV infection in the human subject.

72. The method of claim 71, wherein the administration is to prevent herpes zoster (shingles) in an adult human subject.

73. The method of any one of claims 70-72, wherein the mRNA is administered to the human subject via intramuscular administration, intradermal administration or subcutaneous administration.

74. The method of any one of claims 70-73, wherein the administration is a single dose administration.

75. The method of any one of claims 70-73, wherein the administration comprises multiple doses.

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