RNA formulations and lipids

Abstract

The present disclosure relates to methods of lyophilizing RNA and mixing it with a liquid LNP solution, for example, to create an RNA vaccine or therapeutic, including methods for preparing and administering the vaccine or therapeutic.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to PCT / CN2021 / 136110, filed December 7, 2021, and PCT / CN2022 / 118152, filed September 9, 2022, the entire contents of each of which are incorporated by reference herein for all purposes. [Background technology]

[0003] Messenger RNA (mRNA) is a single-stranded anionic RNA molecule that can affect the expression of desired (disease-specific antigen) proteins once it reaches the cytoplasm of target cells. To date, although RNA has shown great potential for vaccine and therapeutic applications, efficient intracellular delivery of RNA remains a major challenge. Lipid nanoparticles (LNPs) have emerged as the most promising non-viral delivery vehicles, which can encapsulate the delicate RNA, protecting it from degradation, as well as facilitating its intracellular uptake and expression. Currently, the only mRNA products approved by the FDA are mRNA vaccines against COVID-19 (mRNA-1273 and BNT162b2), in which anionic mRNA is encapsulated within LNPs through electrostatic interactions with cationic / ionized lipid components, resulting in LNP-encapsulated mRNA liquid drug products. However, due to the inherent instability of mRNA, LNP-encapsulated mRNA liquid drug products require cold-chain management and storage at ultra-low temperatures, limiting their international use.

[0004] Chickenpox is an acute infectious disease caused by the Varicella-Zoster Virus (VZV). Varicella-Zoster Virus is one of eight herpesviruses known to infect humans and vertebrates. VZV is also known as chickenpox virus, varicella virus, varicella zoster virus, and human herpesvirus type 3 (HHV-3). VZV only infects humans and typically causes chickenpox in children, teenagers, and young adults, and shingles (herpes zoster) in adults (rarely in children). Primary VZV infection, which causes chickenpox (varicella), can lead to complications, including viral or secondary bacterial pneumonia. Even if the clinical symptoms of chickenpox resolve, VZV remains dormant (viral latency) in the nervous system of infected humans in the trigeminal nerve and dorsal root ganglion. In approximately 10-20% of cases, with risk rising to 50% after age 80 (Johnson RW et al., Ther Adv Vaccines, 3:109-120, 2015), VZV reactivates later in life and migrates back from the sensory ganglia to the skin, where it produces a disease (rash) known as herpes zoster or varicella zoster. VZV can also cause a number of neurological conditions, ranging from aseptic meningitis to encephalitis. Other serious complications of VZV infection include postherpetic neuralgia, Mollaley's meningitis, multiple herpes zoster, thrombocytopenia, myocarditis, arthritis, and arterial inflammation in the brain, resulting in stroke, myelitis, ophthalmic herpes, or atypical varicella zoster. Rarely, VZV affects the geniculate ganglion, resulting in lesions along certain branches of the facial nerve. Symptoms can include painful blisters on the tongue and ears, along with facial weakness and hearing loss on one side.

[0005] Currently, there are two approved vaccines for herpes zoster: Zostavax, manufactured by Merck, and Shingrix, manufactured by GSK. Zostavax is a live attenuated virus vaccine. Zostavax is a concentrated version of the vaccine Oka, and was approved by the FDA in the United States in 2006. Zostavax is not recommended for people over 60 years of age because its efficacy decreases with age. It has currently been shown to provide 50% protection for approximately 5 years, after which efficacy gradually decreases over the 5-8 years following vaccination, and its protective efficacy is no longer statistically significant 8 years after vaccination (Morrison VA, et al., Clin Infect Dis, 60:900-909, 2015). GSK's Shingrix employs genetic engineering to express the varicella-zoster virus glycoprotein E (gE) and was approved by the FDA in 2017 for use in people over 50 years of age. Shingrix is ​​a recombinant subunit protein-based vaccine. With a 90% protection rate against herpes zoster, Shingrix reduces the risk of post-herpetic neuralgia and is a preferred alternative to Zostavax. However, the adjuvant used in Shingrix is ​​AS01, manufactured by GSK, which has relatively serious side effects. As one of the components is of natural origin (the bark of Quillaja Saponaria Molina), the use of this adjuvant may also face the problem of limited supply.

[0006] Described herein is a novel VZV vaccine that uses self-replicating RNA encoding VZV viral proteins and triggers potent anti-viral antibody and T cell responses when delivered in vivo. Summary of the Invention

[0007] It has now been discovered that the storage and transportation challenges for RNA vaccines and therapeutics can be overcome by producing LNP-formulated RNA products in a different way, electrostatically adsorbing the RNA to the outside of the LNP, instead of freeze-drying the RNA and encapsulating the freeze-dried RNA when first mixed with the LNP. Following this novel approach, a clinical deployment would in effect involve two vials: one liquid LNP stored at about 2-8°C, and the other freeze-dried RNA stored at about 2-8°C, or even at room temperature. The LNP and RNA contents would be mixed at the bedside just prior to clinical use, making the product "ready to use". This novel approach eliminates the ultra-low temperature cold chain storage required for currently commercially available LNP-encapsulated RNA liquid drug products. Despite the change in product structure, the RNA products developed based on this novel concept are expected to be as safe and effective as their widely evaluated LNP-encapsulated counterparts.

[0008] Described herein are methods for lyophilizing RNA and mixing it with a liquid LNP solution, and uses thereof.

[0009] For example, described herein is a composition or set of compositions that separately comprises each of: (1) a lyophilized polynucleotide composition; and (2) a liquid lipid nanoparticle (LNP) solution.

[0010] In some embodiments, the polynucleotide encodes a protein, polypeptide, or antigen.

[0011] In some embodiments, the polynucleotide comprises at least one of ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA, retroviral RNA, self-replicating RNA (replicon RNA), small interfering RNA (siRNA), microRNA, small nuclear RNA (snRNA), small hairpin (sh)RNA, a riboswitch, a ribozyme, an oligonucleotide, or an aptamer.

[0012] In some embodiments, the polynucleotide comprises RNA, optionally messenger RNA (mRNA) or self-replicating RNA.

[0013] In some embodiments, the RNA is not self-replicating RNA.

[0014] In some embodiments, the RNA is self-replicating RNA.

[0015] In some embodiments, the moisture content of the lyophilized RNA composition is less than 10%.

[0016] In some embodiments, the moisture content of the lyophilized RNA composition is less than 5%.

[0017] In some embodiments, the moisture content of the lyophilized RNA composition is less than 3%.

[0018] In some embodiments, the lyophilized RNA composition is at about 2-8°C.

[0019] In some embodiments, the lyophilized RNA composition comprises at least one suitable protecting agent.

[0020] In some embodiments, suitable protectants include polyols, polymers, sugars, or any combination thereof.

[0021] In some embodiments, a suitable protectant comprises sucrose.

[0022] In some embodiments, the concentration of the protecting agent ranges from 0.5% to 30% by weight of the lyophilized RNA composition.

[0023] In some embodiments, the LNP comprises an ionizable lipid of formula I [ka] wherein R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl R4 and R5 are each independently C1-C 10 is alkyl; R6 and R7 are each independently C1-C 10 Alkyl, C1-C 10 Alkenyl A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; R8 and R9 are each independently C1-C 10 It is an alkyl.

[0024] In some embodiments, L is [ka] or [ka] It is.

[0025] In some embodiments, the LNP comprises an ionizable lipid of formula II [ka] wherein R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18 is an alkyl group Q1 and Q2 are each independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-;-SS-; and R6 and R7 are each independently C1-C 32 It is an alkyl.

[0026] In some embodiments, R and R are each independently C-C 10 It is an alkyl group.

[0027] In some embodiments, R and R are each independently C-C 22 In some embodiments, R and R are each independently C-C 17 It is an alkyl.

[0028] In some embodiments, R1 and R2 are methyl. In some embodiments, Q1 and Q2 are each independently -C(O)-O- or -OC(O)-. In some embodiments, R3 is C3 alkyl. In some embodiments, the LNP comprises: [ka] , [ka] , [ka] , [ka] , [ka] [ka] ; [ka] and [ka] The lipid comprises a lipid selected from the group consisting of:

[0029] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, a structured lipid, and a PEGylated lipid.

[0030] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG 2000). In some embodiments, the LNPs comprise an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG 2000).

[0031] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 30%-70% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0032] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 40%-55% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0033] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 40%-50% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0034] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 45%-50% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0035] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 46%-49% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0036] In some embodiments, the ionizable lipids comprise about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% of the LNP. In some embodiments, the LNPs comprise phospholipids:structural lipids:PEGylated lipids:and ionizable lipids in a molar ratio of 10:40.5:1.5:48.

[0037] In some embodiments, the phospholipid is DOPE or DSPC.

[0038] In some embodiments, the PEGylated lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, or a combination thereof. In some embodiments, the PEGylated lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid, or any combination thereof.

[0039] In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), DMG-PEG 3500, DMG-PEG 5000, DTDAM-PEG 2000 (ALC-0159), DTDAM-PEG 5000, DMG-C-PEG 2000, DMG-C-PEG 5000, DSG-PEG 2000, DSG-PEG 5000, DPG-PEG 2000, DPG-PEG 5000, or any combination thereof.

[0040] In some embodiments, the structured lipid comprises cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, or a combination thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid comprises cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof.

[0041] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionized lipid: 30%-70%, with the sum of the molar ratios of lipids being 100%.

[0042] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid in molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and ionized lipid, with the sum of the lipid molar ratios being 100%, optionally 10:40.5:1.5:48.

[0043] In some embodiments, the LNP comprises DSPC:cholesterol:1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000): and ionized lipid, with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionized lipid: 40%-50%, and the sum of the molar ratios of lipids is 100%.

[0044] In some embodiments, the LNP comprises DSPC:cholesterol:1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000): and ionized lipid, with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionized lipid: 45%-50%, and the sum of the molar ratios of lipids is 100%.

[0045] In some embodiments, the LNP comprises DSPC:cholesterol:1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000): and ionized lipid, with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionized lipid: 46%-49%, and the sum of the molar ratios of lipids is 100%.

[0046] In some embodiments, the ionizable lipid comprises about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% of the LNP. In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionizable lipid in a molar ratio of 10:40.5:1.5:48.

[0047] In some embodiments, the pH of the LNP is 5-6.

[0048] In some embodiments, the particle size of the LNPs is between about 40-300 nm, optionally, the particle size of the LNPs is less than or equal to 160 nm, and optionally, the particle size of the LNPs is between about 140-160 nm.

[0049] In some embodiments, there is a liquid composition comprising lyophilized RNA electrostatically adsorbed to at least one exterior portion of a lipid nanoparticle (LNP) at a molar ratio of ionized lipid:RNA (N / P) of 5:1 to 12:1, optionally at a molar ratio of N / P of 5:1 to 9:1, and optionally at a molar ratio of N / P of 7:1.

[0050] In some embodiments, the LNP comprises an ionizable lipid of formula I.

[0051] In some embodiments, the LNP comprises an ionizable lipid of formula II.

[0052] In some embodiments, the LNP comprises: [ka] , [ka] , [ka] , [ka] , [ka] [ka] ; [ka] and [ka] The lipid composition comprises an ionizable lipid selected from the group consisting of:

[0053] In some embodiments, the LNP comprises an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid.

[0054] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0055] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0056] In some embodiments, the LNP comprises phospholipids:cholesterol:PEGylated lipids: and ionized lipids, with molar ratios ranging from phospholipids: 5%-20%, cholesterol: 18.5%-58.5%, PEGylated lipids: 1%-4%, ionized lipids: 30%-70%, and the sum of the molar ratios of lipids is 100%.

[0057] In some embodiments, the LNP comprises phospholipids:cholesterol:PEGylated lipids: and ionized lipids, with molar ratios ranging from phospholipids: 5%-20%, cholesterol: 18.5%-58.5%, PEGylated lipids: 1%-4%, ionized lipids: 40%-55%, and the sum of the molar ratios of lipids is 100%.

[0058] In some embodiments, the LNP comprises phospholipids:cholesterol:PEGylated lipids: and ionized lipids, with molar ratios ranging from phospholipids: 5%-20%, cholesterol: 18.5%-58.5%, PEGylated lipids: 1%-4%, ionized lipids: 40%-50%, and the sum of the molar ratios of lipids is 100%.

[0059] In some embodiments, the LNP comprises phospholipids:cholesterol:PEGylated lipids:ionized lipids, with molar ratios ranging from phospholipids: 5%-20%, cholesterol: 18.5%-58.5%, PEGylated lipids: 1%-4%, ionized lipids: 45%-50%, and the sum of the molar ratios of lipids is 100%.

[0060] In some embodiments, the LNP comprises phospholipids:cholesterol:PEGylated lipids: and ionized lipids, with molar ratios ranging from phospholipids: 5%-20%, cholesterol: 18.5%-58.5%, PEGylated lipids: 1%-4%, ionized lipids: 46%-49%, and the sum of the molar ratios of lipids is 100%.

[0061] In some embodiments, the ionizable lipid comprises about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% of the LNP. In some embodiments, the LNP comprises phospholipid:cholesterol:PEGylated lipid:and ionizable lipid in a molar ratio of 10:40.5:1.5:48.

[0062] In some embodiments, the phospholipid is DOPE or DSPC.

[0063] In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), DMG-PEG 3500, DMG-PEG 5000, DTDAM-PEG 2000 (ALC-0159), DTDAM-PEG 5000, DMG-C-PEG 2000, DMG-C-PEG 5000, DSG-PEG 2000, DSG-PEG 5000, DPG-PEG 2000, DPG-PEG 5000, or any combination thereof.

[0064] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid in molar ratios ranging from 5%-20% DSPC, 18.5%-58.5% cholesterol, 1%-4% DMG-PEG 2000, and ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0065] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionized lipid: 40%-55%, with the sum of the molar ratios of lipids being 100%.

[0066] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid with molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0067] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid in molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0068] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000:and ionized lipid with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, PEG: 1%-4%, ionized lipid: 46%-49%, with the sum of the molar ratios of lipids being 100%.

[0069] In some embodiments, the ionizable lipid comprises about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% of the LNP. In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionizable lipid in a molar ratio of 10:40.5:1.5:48.

[0070] In some embodiments, the pH of the LNP is 5-6.

[0071] In some embodiments, the particle size of the LNPs is between about 40-300 nm, optionally, the particle size of the LNPs is less than or equal to 160 nm, and optionally, the particle size of the LNPs is between about 140-160 nm.

[0072] In some embodiments, the concentration of RNA is between 10 and 120 μg / mL, optionally between 30 and 60 μg / mL.

[0073] In some embodiments, there is a method for freeze-drying an RNA composition comprising: a) providing a liquid comprising RNA and at least one suitable protectant; b) adding the RNA liquid to a freeze-drying chamber; c) freezing the liquid, the freezing being carried out in four steps at defined temperatures; d) reducing the pressure in the freeze-drying chamber to a pressure below atmospheric pressure; e) drying the frozen liquid to obtain a freeze-dried composition comprising RNA and at least one suitable protectant, optionally with primary and secondary drying steps; and f) equilibrating the pressure in the freeze-drying chamber to atmospheric pressure and removing the freeze-dried composition.

[0074] In some embodiments, suitable protectants include polyols, polymers, sugars, or any combination thereof.

[0075] In some embodiments, the concentration of the protectant ranges from 0.5% to 30% by weight.

[0076] In some embodiments, the pH of the protectant is in the range of 3 to 8, and the total ionic strength is less than or equal to 0.2 mol kg -1 is less than.

[0077] In some embodiments, a suitable protectant comprises 50 mM citrate buffer, pH 6.0, 7.5% (w / v) sucrose.

[0078] In some embodiments, 1-150 μg / mL of RNA liquid is added to the lyophilization chamber.

[0079] In some embodiments, the first freezing step freezes the RNA to 25° C. to 2° C. with full cooling capacity.

[0080] In some embodiments, the second freezing step freezes the RNA at 2° C. for 1 hour.

[0081] In some embodiments, the third freezing step freezes the RNA to 2° C. to −50° C., optionally with full cooling capacity.

[0082] In some embodiments, the fourth freezing step freezes the RNA at -50°C for 3 hours or more.

[0083] In some embodiments, the pressure in the lyophilization chamber is reduced to 0.01 mbar.

[0084] In some embodiments, the first primary drying step dries the RNA at a temperature between -50°C and -47°C for 1 hour.

[0085] In some embodiments, the second primary drying step dries the RNA at -47°C for 20 hours.

[0086] In some embodiments, the third primary drying step dries the RNA at -47°C to -20°C for 3 hours.

[0087] In some embodiments, the fourth primary drying step dries the RNA at -20°C for 1 hour.

[0088] In some embodiments, the pressure in the freeze-drying chamber is increased to 0.02 mbar.

[0089] In some embodiments, the first secondary drying step dries the RNA at -22°C to 4°C for 3 hours.

[0090] In some embodiments, the second secondary drying step dries the RNA at 4° C. for 2 hours.

[0091] In some embodiments, the pressure in the freeze-drying chamber is increased to 0.08 mbar.

[0092] In some embodiments, the third secondary drying step dries the RNA at 4° C. to 30° C. for 4 hours.

[0093] In some embodiments, the fourth secondary drying step dries the RNA at 30° C. for 4 hours.

[0094] In some embodiments, the moisture content of the lyophilized RNA is less than 10%.

[0095] In some embodiments, the moisture content of the lyophilized RNA is less than 5%.

[0096] In some embodiments, the moisture content of the lyophilized RNA is less than 3%.

[0097] In some embodiments, the lyophilized RNA is at 2-8°C.

[0098] In some embodiments, the lyophilized RNA is mixed with LNPs.

[0099] In some embodiments, the LNP is a liquid solution added to lyophilized RNA.

[0100] In some embodiments, the lyophilized RNA and the LNPs are mixed at room temperature.

[0101] In some embodiments, the lyophilized RNA and the LNPs are mixed prior to administration.

[0102] In some embodiments, there is a method of making the composition of any of the embodiments, comprising mixing lyophilized RNA with LNPs.

[0103] In some embodiments, the LNPs are a liquid solution and are added to the lyophilized RNA.

[0104] In some embodiments, the lyophilized RNA and the LNPs are mixed at room temperature.

[0105] In some embodiments, the lyophilized RNA and the LNPs are mixed prior to administration.

[0106] In some embodiments, the amount of LNP added to the lyophilized RNA results in a molar ratio of ionizable lipid:RNA (N:P) ranging from 2:1 to 12:1, optionally 5:1 to 9:1, and optionally 7:1.

[0107] In some embodiments, the RNA concentration in the composition to be mixed is 10 to 120 μg / mL, optionally 30 to 60 μg / mL.

[0108] In some embodiments, the RNA is electrostatically adsorbed to at least one exterior portion of the LNP.

[0109] In some embodiments, the method comprises administering to a subject a composition of any of the embodiments.

[0110] In some embodiments, the composition is administered to the subject intramuscularly, intravenously, intraarterially, intradermally, subcutaneously, intraperitoneally, intraventricularly, or intracranially.

[0111] In some embodiments, administering the composition enhances an immune response in the subject.

[0112] In some embodiments, the composition is administered to the subject at least twice.

[0113] In some embodiments, the composition is administered to the subject about 1-8 weeks after the first administration.

[0114] In some embodiments, the composition is administered to a subject at a dose of 1-100 μg, optionally 8, 10 μg, or 30 μg of RNA.

[0115] In some embodiments, a kit includes an RNA composition according to any of the embodiments, a liquid LNP solution composition according to any of the embodiments, and instructions for use.

[0116] In some embodiments, the set of compositions is stored in separate glass vials.

[0117] In some embodiments, the liquid LNP solution composition is at a temperature of 2-8°C.

[0118] In some embodiments, the lyophilized RNA composition is stored at room temperature.

[0119] In some embodiments, the lyophilized RNA composition is stored at a temperature between 2 and 8°C.

[0120] As used herein, lipids of formula I: [ka] or a pharma- ceutically acceptable salt thereof is provided, During the ceremony, R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl; R4 and R5 are each independently C1-C 10 is alkyl; R6 and R7 are each independently C1-C 10 Alkyl, or C1-C 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; R8 and R9 are each independently C1-C 30 It is an alkyl.

[0121] In some embodiments, L is [ka] or [ka] It is.

[0122] As used herein, a lipid of formula II: [ka] or a pharma- ceutically acceptable salt thereof is provided, During the ceremony, R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18 is alkyl; Q1 and Q2 are each independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-; -SS-; R6 and R7 are each independently C 18 -C 32 It is an alkyl. In some embodiments, R1 and R2 are methyl. In some embodiments, Q1 and Q2 are each independently -C(O)-O- or -OC(O)-. In some embodiments, R3 is C3 alkyl.

[0123] In some embodiments, the lipid is [ka] [ka] [ka] ; [ka] and [ka] is selected from the group consisting of:

[0124] In some embodiments, provided herein is a pharma- ceutically acceptable composition comprising any of the lipids of the above embodiments and a pharma- ceutically acceptable carrier. In some embodiments, an ionizable lipid of formula I: [ka] Provided herein is a lipid nanoparticle (LNP) comprising: During the ceremony, R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl; R4 and R5 are each independently C1-C 10 is alkyl; R6 and R7 are each independently C1-C 10 Alkyl, C1-C 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; and R8 and R9 are each independently C1-C 30 It is an alkyl.

[0125] In some embodiments, L is [ka] or [ka] It is.

[0126] In some embodiments, the LNP comprises an ionizable lipid of formula II: [ka] Including, During the ceremony, R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18 is an alkyl group Q1 and Q2 are each independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-;-SS-; and R6 and R7 are each independently C1-C 32 It is an alkyl.

[0127] In some embodiments, R and R are each independently C-C 22 It is an alkyl.

[0128] In some embodiments, R and R are each independently C-C 10 It is an alkyl group.

[0129] In some embodiments, R and R are each independently C-C 17 It is an alkyl.

[0130] In some embodiments, the LNP comprises: [ka] , [ka] , [ka] , and [ka] The lipid comprises a lipid selected from the group consisting of:

[0131] In some embodiments, R1 and R2 are methyl.

[0132] In some embodiments, Q1 and Q2 are each independently -C(O)-O- or -OC(O)-.

[0133] In some embodiments, R3 is a straight chain C3 alkyl.

[0134] In some embodiments, the LNP comprises: [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , [ka] , and [ka] The lipid comprises a lipid selected from the group consisting of:

[0135] In some embodiments, the LNP further comprises a phospholipid, a structured lipid, and a PEGylated lipid.

[0136] In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids, with molar ratios ranging from 5%-20% phospholipids, 18.5%-58.5% structural lipids, 1%-4% PEGylated lipids, and 30%-70% ionized lipids, and the sum of the molar ratios of lipids is 100%.

[0137] In some embodiments, the LNP comprises DSPC:cholesterol:1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000): and ionizable lipid, with molar ratios ranging from DSPC: 5%-20%, cholesterol: 30%-55%, DMG-PEG 2000: 1%-4%, ionizable lipid: 30%-70%, with the sum of the lipid molar ratios being 100%, and optionally, the molar ratios being 10:40.5:1.5:48.

[0138] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0139] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and a PEGylated lipid, optionally 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0140] In some embodiments, the pH of the LNP is 5-6.

[0141] In some embodiments, the particle size of the LNPs is about 40-300 nm, optionally, the particle size of the LNPs is less than 160 nm, optionally, the particle size of the LNPs is about 140-160 nm, and optionally, the particle size of the LNPs is about 90-120 nm.

[0142] In some embodiments, the LNPs are stable when stored at 2-8°C.

[0143] In some embodiments, the change in average size of the LNPs is less than 40 nm when stored at 2-8° C. for 7 days or more, 14 days or more, or 30 days or more. In some embodiments, the change in average size of the LNPs is less than 30 nm, less than 20 nm, or optionally less than 10 nm.

[0144] In some embodiments, provided herein are methods of using any of the LNPs described above to deliver a polynucleotide to a cell for expression of a polypeptide of interest. In some embodiments, the polynucleotide is electrostatically adsorbed to at least one exterior portion of the LNP or is encapsulated within the LNP.

[0145] In some embodiments, the polynucleotide is protected and reduced from degradation during delivery to a cell.

[0146] In some embodiments, the polynucleotide is in lyophilized form prior to mixing with the LNPs.

[0147] In some embodiments, the polynucleotide is in liquid form prior to mixing with the LNPs.

[0148] Described herein is a self-replicating RNA (srRNA) that includes a nucleotide sequence encoding a Varicella-Zoster Virus (VZV) antigen.

[0149] In some embodiments, the VZV antigen comprises a VZV glycoprotein E (gE) antigen.

[0150] In some embodiments, the gE antigen comprises a VZV Oka strain gE protein.

[0151] In some embodiments, the gE antigen comprises a mature extracellular domain sequence of a gE antigen, or an immunogenic fragment thereof.

[0152] In some embodiments, the sequence of the extracellular domain of the gE antigen comprises the sequence shown in SEQ ID NO:3.

[0153] In some embodiments, the nucleotide sequence encoding a VZV antigen is operably linked to a promoter.

[0154] In some embodiments, the srRNA comprises a 5' cap untranslated region (UTR), one or more nonstructural genes, a promoter, and a 3' terminal polyadenylation (polyA) region.

[0155] In some embodiments, the one or more nonstructural genes comprise four nonstructural genes (nsp1-4) and the promoter comprises a 26S subgenomic promoter.

[0156] In some embodiments, the nucleotide sequence encoding a VZV antigen is operably linked to a promoter.

[0157] In some embodiments, the srRNA comprises, from 5' to 3', a 5'UTR, one or more nonstructural genes, a promoter, a nucleotide sequence encoding a VZV antigen, and a 3' polyA region.

[0158] In some embodiments, the srRNA lacks one or more nucleotide sequences encoding one or more structural protein sequences, and optionally, a nucleotide sequence encoding a VZV antigen is inserted in place of one or more nucleotide sequences encoding one or more structural protein sequences.

[0159] In some embodiments, the srRNA is TC-83 VEEV srRNA.

[0160] In some embodiments, the srRNA sequence comprises the sequence shown in SEQ ID NO:4.

[0161] In some embodiments, the srRNA comprises an mRNA cap.

[0162] In some embodiments, the mRNA cap comprises m7G (cap 0), m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated) (cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or an anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated).

[0163] In some embodiments, the srRNA is lyophilized.

[0164] In some embodiments, the lyophilized srRNA is at a temperature of 22°C or below, optionally, about 2-8°C.

[0165] In some embodiments there is a composition comprising an srRNA according to any of the above claims and a delivery vehicle.

[0166] In some embodiments, the delivery vehicle comprises a lipid nanoparticle (LNP).

[0167] In some embodiments, the LNPs comprise an ionizable lipid, heptadecan-9-yl 8-(3-(((4-(dimethylamino)butanoyl)oxy)methyl)-4-((8-(nonyloxy)-8-oxooctyl)oxy)phenoxy)octanoate. In some embodiments, the LNP comprises an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000).

[0168] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMG-PEG2000).

[0169] In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and a PEGylated lipid comprising a polyethylene glycol moiety.

[0170] In some embodiments, the srRNA is adsorbed to the surface of the LNP.

[0171] In some embodiments, the composition comprises a molar ratio of DSPC:cholesterol:PEG:ionized lipid of 5:20:0:20 to 25:70:5:60, optionally 10:48:2:40, with an N:P (lipid:srRNA) ratio ranging from 2:1 to 12:1, optionally 8:1.

[0172] In some embodiments, the composition comprises a molar ratio of DOPE:cholesterol:PEG:ionized lipid of 5:20:0:20 to 25:70:5:60, optionally 10:48:2:40, with an N:P (lipid:srRNA) ratio ranging from 2:1 to 12:1, optionally 8:1.

[0173] In some embodiments, the particle size of the composition is about 40-300 nm.

[0174] In some embodiments, the composition enhances an immune response in a subject after administration.

[0175] In some embodiments, the immune response comprises an antigen-specific adaptive immune response.

[0176] In some embodiments, the adaptive immune response comprises B cells, CD4+ T cells, and / or CD8+ T cells.

[0177] In some embodiments, the adaptive immune response comprises CD8+ T cells.

[0178] In some embodiments, the composition lacks a separate adjuvant component.

[0179] In some embodiments, there is a method of enhancing an immune response to VZV in a subject, comprising administering to the subject a composition of any of the embodiments.

[0180] In some embodiments, there is a method of preventing or treating herpes zoster or a VZV-associated disease in a subject, comprising administering to the subject a composition described in the embodiments.

[0181] In some embodiments, the composition is administered to the subject intramuscularly.

[0182] In some embodiments, the composition is administered to the subject about 4 weeks after the first administration.

[0183] In some embodiments, the composition is administered to the subject at least twice.

[0184] In some embodiments, the composition is administered to a subject in a dose of 1-100 μg, optionally, 10 μg or 30 μg.

[0185] In some embodiments, the composition enhances an immune response in a subject after administration.

[0186] In some embodiments, the immune response comprises an antigen-specific adaptive immune response.

[0187] In some embodiments, the adaptive immune response comprises B cells, CD4+ T cells, and / or CD8+ T cells.

[0188] In some embodiments, the adaptive immune response comprises CD8+ T cells.

[0189] In some embodiments, there is a method of making a composition of any of the embodiments, comprising mixing srRNA with a delivery vehicle.

[0190] In some embodiments, the srRNA is optionally lyophilized at a temperature between 2 and 8°C.

[0191] In some embodiments, the delivery vehicle is an LNP, and optionally is in a liquid state, optionally at a temperature of 2-8°C.

[0192] In some embodiments, there is a kit comprising an srRNA as described in any of the embodiments, a delivery vehicle, and instructions for use.

[0193] In some embodiments, the srRNA of the kit is lyophilized, optionally at a temperature of 2-8°C.

[0194] In some embodiments, the delivery vehicle of the kit is an LNP, and optionally is in a liquid state, optionally at a temperature of 2-8°C.

[0195] In some embodiments, described herein is a composition or set of compositions that separately comprises each of: (1) a lyophilized polynucleotide composition; and (2) a liquid lipid nanoparticle (LNP) solution.

[0196] In some embodiments, the LNP comprises an ionizable lipid of formula II: [ka] Including, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18each of Q1 and Q2 is independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-; -SS-; and each of R6 and R7 is independently a C1-C 32 In some embodiments, R and R are each independently a C-C alkyl group. 10 alkyl group; and R and R are each independently a C-C 17 In some embodiments, R and R are methyl, Q and Q are each independently -C(O)-O- or -OC(O)-, and R is a straight chain C alkyl.

[0197] In some embodiments, the ionizable lipid is [ka] It is.

[0198] In some embodiments, the lyophilized polynucleotide composition comprises a self-replicating RNA. In some embodiments, the lyophilized polynucleotide encodes a protein, a polypeptide, or an antigen. In some embodiments, the lyophilized polynucleotide comprises at least one of messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), viral RNA, retroviral RNA, self-replicating RNA (replicon RNA), small interfering RNA (siRNA), microRNA, small nuclear RNA (snRNA), small hairpin (sh)RNA, riboswitch, ribozyme, oligonucleotide, or aptamer. In some embodiments, the lyophilized polynucleotide is a self-replicating RNA. The composition of claim 1, wherein the lyophilized polynucleotide is not a self-replicating RNA. In some embodiments, the moisture content of the lyophilized polynucleotide composition is less than 5%. In some embodiments, the lyophilized polynucleotide composition is stored at about 2-8°C. In some embodiments, the lyophilized polynucleotide composition comprises at least one suitable protective agent. In some embodiments, the suitable protective agent comprises a polyol, a polymer, a sugar, or any combination thereof. In some embodiments, a suitable protectant comprises sucrose, hi some embodiments, the concentration of the protectant ranges from 0.5% to 30% by weight of the lyophilized polynucleotide composition.

[0199] In some embodiments, the LNP comprises an ionizable lipid of formula I: [ka] Including, wherein R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl; R4 and R5 are each independently C1-C 10alkyl; R and R are each independently C-C 10 Alkyl, C1-C 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; and R8 and R9 are each independently C1-C 30 In some embodiments, L is [ka] or [ka] wherein the CH2 moiety is attached to Q1.

[0200] In some embodiments, the ionizable lipid is [ka] , [ka] , and [ka] is selected from the group consisting of:

[0201] In some embodiments, the ionizable lipid is [ka] , [ka] , [ka] , and [ka] is selected from the group consisting of:

[0202] In some embodiments, the LNPs comprise an ionizable lipid, a phospholipid, a structured lipid, and a PEGylated lipid. In some embodiments, the LNPs comprise an ionizable lipid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and a PEGylated lipid. In some embodiments, the LNPs comprise an ionizable lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and a PEGylated lipid. In some embodiments, the PEGylated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DMG-PEG 2000). In some embodiments, the LNP comprises phospholipids: structural lipids: PEGylated lipids: and ionized lipids in molar ratios ranging from 5% to 20% phospholipids, 18.5% to 58.5% structural lipids, 1% to 4% PEGylated lipids, and 30% to 70% ionized lipids, where the sum of the lipid molar ratios is 100%. In some embodiments, the LNP comprises DSPC: cholesterol: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000): and ionized lipids in molar ratios ranging from 5% to 20% DSPC, 30% to 55% cholesterol, 1% to 4% DMG-PEG 2000, and 30% to 70% ionized lipids, where the sum of the lipid molar ratios is 100%. In some embodiments, the pH of the LNP solution is 5 to 6. In some embodiments, the LNPs have a particle size of about 40-300 nm, optionally, the LNPs have a particle size of less than or equal to 160 nm, optionally, the LNPs have a particle size of about 140-160 nm, and optionally, the LNPs have a particle size of about 90-120 nm. In some embodiments, the lyophilized polynucleotide composition comprises SEQ ID NO:4.

[0203] In some embodiments, described herein is a self-replicating RNA (srRNA) vector comprising, in 5' to 3' order: a) a Cap1 cap; b) a 5'UTR; c) one or more structural genes; d) a gene of interest (GOI); e) a 3'UTR having about 180 to about 400 nucleotides; and f) a polyA tail having about 60 to about 100 nucleotides. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least about 80% identity to the nucleic acid sequence SEQ ID NO:6. In some embodiments, the srRNA vector comprises at least 1.5X, 2.0X, 2.5X, 3.0X, or more GOI expression compared to an unmodified vector. In some embodiments, the srRNA vector comprises at least 2X, 3X, 4X, 5X, 6X, or more improved immune response compared to an unmodified vector. In some embodiments, the GOI is a Varicella-Zoster Virus (VZV) antigen. In some embodiments, the VZV antigen comprises a VZV glycoprotein E (gE) antigen.

[0204] In some embodiments, a self-replicating RNA (srRNA) is described herein that includes a nucleotide sequence encoding a Varicella-Zoster Virus (VZV) antigen. In some embodiments, the VZV antigen includes a VZV glycoprotein E (gE) antigen. In some embodiments, the gE antigen includes a VZV Oka strain gE protein. In some embodiments, the gE antigen includes a mature extracellular domain sequence of the gE antigen, or an immunogenic fragment thereof. In some embodiments, the sequence of the extracellular domain of the gE antigen includes SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding the VZV antigen is operably linked to a promoter. In some embodiments, the srRNA includes a 5' cap untranslated region (UTR), one or more nonstructural genes, a promoter, and a 3' terminal polyadenylation (polyA) region. In some embodiments, the one or more nonstructural genes include four nonstructural genes (nsp1-4), and the promoter includes a 26S subgenomic promoter. In some embodiments, the srRNA comprises, from 5' to 3', a 5'UTR, one or more nonstructural genes, a promoter, a nucleotide sequence encoding a VZV antigen, and a 3' polyA region. In some embodiments, the srRNA lacks one or more nucleotide sequences encoding one or more structural protein sequences, optionally wherein a nucleotide sequence encoding a VZV antigen is inserted in place of one or more nucleotide sequences encoding one or more structural protein sequences. In some embodiments, the srRNA is a TC-83 VEEV srRNA. In some embodiments, the srRNA sequence comprises SEQ ID NO:2. In some embodiments, the srRNA sequence comprises SEQ ID NO:4. In some embodiments, the srRNA comprises a polyA tail having a length of 30-100 nucleotides. In some embodiments, the srRNA comprises a 3'UTR having a length of 100-400 nucleotides. In some embodiments, the srRNA comprises an mRNA cap.In some embodiments, the mRNA cap comprises m7G (cap0), m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated) (cap1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated). In some embodiments, the srRNA comprises a Cap1 cap, a VZV glycoprotein E (gE) antigen, a 3'UTR having a length of about 270 nucleotides, and a poly-A tail having a length of about 65 nucleotides.

[0205] In some embodiments, described herein is a composition or set of compositions comprising each of: (1) a lyophilized polynucleotide composition; and (2) a liquid lipid nanoparticle (LNP) solution, wherein the lyophilized polynucleotide is a self-replicating RNA and the LNP has the formula: [ka] Contains ionized lipids.

[0206] In some embodiments, the lyophilized polynucleotide comprises a polyA tail between 30 and 100 nucleotides in length. In some embodiments, the lyophilized polynucleotide comprises a cap structure selected from the group consisting of m7G (cap 0), m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated) (cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated). In some embodiments, the lyophilized polynucleotide comprises a nucleotide sequence encoding a Varicella-Zoster Virus (VZV) antigen. In some embodiments, the lyophilized polynucleotide comprises a VZV glycoprotein E (gE) antigen. In some embodiments, the lyophilized polynucleotide comprises a 3'UTR between 180 and 400 nucleotides in length. In some embodiments, the lyophilized polynucleotide comprises a Cap1 cap, a VZV glycoprotein E (gE) antigen, a 3'UTR having a length of about 270 nucleotides, and a polyA tail having a length of about 65 nucleotides. In some embodiments, the lyophilized polynucleotide comprises a Cap1 cap, a SARS-CoV-2 spike protein (RBD), a 3'UTR having a length of about 270 nucleotides, and a polyA tail having a length of about 65 nucleotides.

[0207] In some embodiments, described herein is a self-replicating RNA (srRNA) comprising SEQ ID NO:4 that encodes the Varicella-Zoster Virus (VZV) gE antigen. [Brief description of the drawings]

[0208] [Figure 1A] 1A-1B show the structures of ionized lipids used to test the "ready-to-use" RNA formulations. [Figure 1B] 1A-1B show the structures of ionized lipids used to test the "ready-to-use" RNA formulations. [Diagram 2] Western blot of Vero E6 cell line samples transfected with either "ready to use" or LNP-encapsulated S-2P RNA probed against the SARS-CoV-2 spike glycoprotein. [Diagram 3] Figure 1 shows the treatment protocol for BALB / c mice vaccinated with either "ready-to-use" or LNP-encapsulated S-2P RNA encoding the spike glycoprotein of SARS-CoV-2. Arrows indicate blood sampling days. [Figure 4] Geometric mean titers are shown for BALB / c mice vaccinated with either "ready-to-use" or LNP-encapsulated S-2P RNA encoding the spike glycoprotein of SARS-CoV-2 on the days indicated. [Diagram 5] 1 shows a treatment protocol with srRNA vaccine for cynomolgus monkey studies. [Figure 6] VZV gE-specific antibody titers in serum of cynomolgus macaques are shown on the indicated days. Arrows indicate doses of srRNA vaccine. [Figure 7] Antibody binding activity in serum of immunized cynomolgus monkeys on the days indicated is shown. [Figure 8] 1 shows individual cytokine expression levels at day 90 in peripheral blood mononuclear cells from cynomolgus monkeys immunized with 10 μg or 30 μg of srRNA vaccine. [Figure 9] 1 shows an exemplary construction of an srRNA encoding the VZV glycoprotein E antigen. [Figure 10] Figures 10A and 10B show the relative potency of selected lyophilized RNA stored at the indicated temperatures and at the indicated time points, tested using an in vitro potency assay that compares the overall intensity of the fluorescent signal against a reference standard. [Figure 11] 1 shows a VZV srRNA vaccine treatment protocol for mouse studies. [Figure 12]Serum antibody levels in immunized mice as determined by ELISA are shown. Arrows below the graph indicate time of VZV srRNA vaccination. Each data point shown is the mean EC50 in individual mouse sera in each group (n=12 for groups G1-G6, n=8 for group G7). Bars indicate geometric mean ± geometric standard deviation (SD). [Figure 13] Figures 13A and 13B show the frequencies of antigen-specific T cell subsets measured from splenocytes of immunized mice after in vitro restimulation with peptide pools spanning the VZV gE protein. Bars represent individual means in each group, with mean ± SD. (**, p<0.01). [Figure 14] Figure 1 shows the frequency of cytokine-expressing VZV gE-specific T cells measured by flow cytometry. Results of CD4+ T cell immune responses. Bars represent individual means in each group, with mean ± SD. (**, p<0.01 vs. G5 or G7). [Figure 15] 1 shows an exemplary construction of a vector structure with an extended 3'UTR and / or polyA tail. [Figure 16] 1 shows an exemplary srRNA structure. [Figure 17A] Western blots probed for RBD expression are shown. [Figure 17B] Western blot relative RBD expression across samples is shown. [Figure 18] 1 shows in vivo BLI imaging of mice treated with Structure 1, Structure 3, Structure 4, or vehicle. [Figure 19] Quantification of total flux in treated mice is shown in Figure 18. Significant differences between Structure 1 group and other groups are indicated by a vertical asterisk atop the other asterisks (*p<0.05, **p<0.01). [Figure 20A] The immunization schedule for mice in the experiment in FIG. 20B is shown. [Figure 20B]Quantification of total IgG antibody titers in mice vaccinated with a dual antigen VZV gE srRNA vaccine is shown. [Figure 21] A and B show the frequency of cytokine-expressing VZV gE-specific T cells measured by flow cytometry. Figure 21A shows the results of CD4+ T cell immune responses. Figure 21B shows the results of CD8+ T cell immune responses. Bars represent individual means in each group, with mean ± SD. (*p<0.05, **p<0.01). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0209] Freeze-drying of RNA The present disclosure provides methods for lyophilizing RNA. The present disclosure further relates to lyophilized compositions obtainable by the methods provided herein, as well as pharmaceutical compositions and kits containing the lyophilized compositions.

[0210] Both DNA and RNA nucleic acids have been used in vaccines, either in naked or complexed form. RNA presents several superior properties over DNA, so the application of RNA in vaccines or other therapeutic agents represents a preferred tool in modern molecular medicine. Transfection of DNA molecules can lead to complications. For example, applying DNA molecules carries the risk of DNA integration into the host genome. Integration of foreign DNA into the host genome can affect the expression of host genes, triggering the expression of oncogenes or the inactivation of tumor suppressor genes. Furthermore, essential genes, and consequently the products of such essential genes, can also be inactivated by integration of foreign DNA into the coding region of the gene. The consequences of such an event can be particularly dangerous when DNA is integrated into a gene involved in the control of cell proliferation. Despite the risks associated with its application, DNA still represents an important tool. However, these risks do not arise when RNA is used instead of DNA.

[0211] The advantage of using RNA rather than DNA is that viral promoter elements do not need to be administered in vivo and cannot integrate into the genome, and RNA does not need to overcome the nuclear barrier to exert its function.

[0212] However, the main drawback of using RNA is its instability. Although it is understood that DNA, such as naked DNA, is usually not stable when introduced into the circulatory system of a patient and therefore may have little chance of affecting most disease processes (see Poxon et al., Pharmaceutical development and Technology, 5(1), 115-122 (2000)), the stability problem is more pronounced in the case of RNA. It is generally known that the physicochemical stability of RNA molecules in solution is low. RNA is susceptible to hydrolysis by ubiquitous ribonucleases or by divalent cations and is typically degraded rapidly, for example, already after a few hours or days in solution. For example, when RNA is stored in solution at room temperature for a few hours or days, rapid degradation occurs even in the absence of RNases. To avoid such rapid degradation, RNA (in solution) is typically stored at -20°C, or even at -80°C, and under RNase-free conditions to prevent RNA degradation. However, such storage conditions do not adequately prevent loss of functionality over time. Moreover, applying such conditions is very costly, especially in transportation and storage, whenever such low temperatures need to be guaranteed.

[0213] One method of increasing the stability of RNA involves lyophilization, or freeze-drying, of RNA. Lyophilization is a globally known and recognized method used to improve the storage stability of temperature-sensitive biomolecules. During lyophilization, solvents such as water are typically removed from the frozen sample by sublimation.

[0214] The process of freeze-drying is usually characterized by a primary and a secondary drying step. During the primary drying step, the free, i.e. unbound, water surrounding the biomolecules and, optionally, further components, evaporates from the frozen solution. The water bound by the biomolecules by molecular mechanisms can then be removed in the secondary drying step by applying thermal energy. In both cases, the hydration sphere around the biomolecules is lost.

[0215] During freeze-drying, a sample containing biomolecules is first cooled below the freezing point of the solution, resulting in the freezing of the water contained therein. Depending on the temperature, the cooling rate (freezing rate) and the time for freezing, among other parameters, crystals may form. This stresses the biomolecules and other components of the solution, which in the case of nucleic acids may lead to damage to the biomolecules, such as strand breakage, disruption of the supercoil structure, etc. Furthermore, the reduction in volume and loss of hydration spheres may impart autocatalytic decomposition processes, for example with traces of transition metals. In addition, traces of acid and base concentrations may result in significant changes in the pH value. Freeze-drying involves two types of stress: freezing and drying. Both types of stress are known to damage nucleic acids, such as non-viral vectors or plasmid DNA. In the literature, a large number of cryoprotectants and cryoprotectants are discussed for freeze-drying purposes to prevent these damages. In this context, cryoprotectants are understood as excipients that can affect the structure of the ice and / or the eutectic or glass transition temperature of the mixture. Cryoprotectants are typically excipients that partially or completely replace the hydration sphere around the molecule, and thus can at least partially prevent catalytic and hydrolytic processes.

[0216] In the specific context of DNA, freeze-drying causes the removal of a sphere of hydration around the DNA where there appear to be approximately 20 hydrogen molecules per most tightly bound nucleotide pair. These water molecules do not form ice-like structures upon cryo-cooling. At 0% relative humidity, dehydrating DNA in the presence of hygroscopic salts leaves only 5 or 6 water molecules (Tao et al., Biopolymers, 28, 1019-1030 (1989)).

[0217] Although freeze-drying can increase the stability of DNA in long-term storage, some damage can also be caused by the initial freeze-drying process, potentially by changes in DNA secondary structure, breakage of nucleic acid strands, or concentration of reactive elements, such as contaminating metals. Freeze-drying can also cause damage during the initial freeze-drying process in other nucleic acids, such as RNA. Agents that can substitute for non-frozen water, such as some carbohydrates, can exhibit cryoprotective properties for DNA and other molecules during freeze-drying of intact bacteria (Israeli et al, Cryobiology, 30, 519-523 (1993); or Rudolph et al, Arch. Biochem. Biophys., 245, 134-143 (1986)). During freeze-drying, certain carbohydrates, such as some sugars, seem to play a central role in stabilizing nucleic acid molecules. However, when using cryoprotectants and cryoprotectants, general rules cannot be applied due to the effects on different groups of compounds. Therefore, cryoprotectants are used herein.

[0218] Cryoprotective properties have been described specifically for sucrose, glucose, and trehalose. These make it possible, at least in part, to recover the transfection efficiency that is often lost after lyophilization (Maitani et al., 2008, supra; Yadava, P., M. Gibbs, et al. (2008). AAPS PharmSciTech 9(2):335-41; Werth, S., B. Urban-Klein, et al. (2006), J Control Release 112(2):257-70; Brus, C, E. Kleemann, et al. (2004), J Control Release 95(1):119-31; Poxon, SW and JA Hughes (2000), Pharm Dev Technol 5(1):115-22; Anchordoquy, TJ, JF Carpenter, et al. (1997), Arch Biochem Biophys 348(1):199-206).

[0219] Sugars may prevent loss of activity due to the lyophilization process, primarily by preventing particle fusion / aggregation, especially in the case of liposome-complexed nucleic acids (Yadava et al, 2008, supra; Katas, H., S. Chen, et al. (2008), J Microencapsul:1-8; Molina et al, supra, 2001). In particular, Poxon et al. (2000, supra) investigated the effect of lyophilization on plasmid DNA activity. Poxon et al. (2000, supra) hypothesized that damage to plasmid DNA was indicated by a change in DNA structure from supercoiled to open circular and linear forms. However, the percentage of supercoiled DNA did not change after lyophilization and subsequent DMED treatment, suggesting that other effects were responsible for the loss of transfection efficiency. Poxon et al. (2000, supra) found that the loss of plasmid DNA activity, as measured in an in vitro transfection assay, could be ameliorated by using carbohydrates during lyophilization of the plasmid DNA. Glucose (a monosaccharide), sucrose, and lactose (a disaccharide) were used as cryoprotectants. However, Poxon et al. (2000, supra) only conducted studies with plasmid DNA.

[0220] Although a large amount of prior art literature has suggested stabilizing nucleic acids during lyophilization in the context of plasmid DNA, only a few publications have focused on stabilizing other nucleic acids, such as RNA, during lyophilization and long-term storage, and furthermore, lyophilization under controlled conditions has hardly been described for RNA.

[0221] Therefore, it is an object of the present disclosure to provide a method for freeze-drying RNA that is scalable, reproducible, and applicable to the manufacture of pharmaceutical products. In particular, it is an object of the present disclosure to provide a method for freeze-drying RNA that preferably maintains the integrity and biological activity of the RNA. It is a further object of the present disclosure to provide a composition comprising RNA that is suitable for storage at ambient temperature and for long periods of time.

[0222] The present disclosure solves these problems presented by the claimed subject matter. The present disclosure provides a method for freeze-drying RNA. In particular, the present disclosure provides a method for freeze-drying RNA, the method comprising the steps of: Providing a liquid comprising RNA and at least one suitable protection agent; adding the RNA liquid to a freeze-drying chamber; Freezing the liquid, said freezing being carried out in four steps at defined temperatures; reducing the pressure in the freeze-drying chamber to subatmospheric pressure; drying the frozen liquid to obtain a lyophilized composition comprising RNA and at least one suitable protecting agent, said drying step comprising a primary and a secondary drying step; allowing the pressure in the lyophilization chamber to equilibrate to atmospheric pressure and removing the lyophilized composition; The present invention teaches a method including:

[0223] In certain embodiments, steps (a)-(f) are performed in the order described above. In some embodiments, they may be performed in an alternative order. In some embodiments, the single steps may be performed simultaneously or may overlap. The method may be suitable for use on an industrial scale.

[0224] The method according to the present disclosure can be used to produce a composition comprising RNA in a reproducible manner. The composition comprising RNA according to the present disclosure can be freely stored, transported, and applied, for example, in the medical field (e.g., as a vaccine or therapeutic agent) without the use of freezing agents, while maintaining high integrity and biological activity of the RNA in the composition.

[0225] In some embodiments, the lyophilized compositions obtainable by the disclosed methods are characterized by a residual water content, preferably in the range of about 0.1% (w / w) to about 10% (w / w), optionally about 1% (w / w) to about 8% (w / w), optionally about 2% (w / w) to about 5% (w / w), optionally about 2% (w / w) to about 4%, optionally about 3% (w / v), optionally less than 3% (w / v), based on the total weight of the lyophilized composition.

[0226] In some embodiments, the liquid provided in step a) of the present disclosure comprises at least one cryoprotectant, and the cryoprotectant is a carbohydrate. Such carbohydrates can include any carbohydrate suitable for preparing pharmaceutical compositions, and are preferably, but not limited to, monosaccharides, such as glucose, fructose, sucrose, galactose, sorbose, mannose, and mixtures thereof; disaccharides, such as lactose, maltose, sucrose, trehalose, cellobiose, and mixtures thereof; polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, dextrin, cellulose, starch, and mixtures thereof; and alditols, such as glycerol, mannitol, xylitol, maltitol, lactitol, xylitol sorbitol, pyranosyl sorbitol, myo-inositol, and mixtures thereof. Preferred examples of sugars that can be included in the liquid provided in step a) of the present disclosure include lactose, mannose, mannitol, sucrose, or trehalose. Generally, preferred sugars in this context have a high water displacement activity and a high glass transition temperature. Furthermore, sugars suitable for use in the liquid provided in step a) of the present disclosure are preferably not hygroscopic and are hydrophilic. In addition, the sugar preferably has a low tendency to crystallize.

[0227] In some embodiments, the liquid provided in step a) of the present disclosure comprises at least 0.01% (w / w), preferably at least 0.1% (w / w), at least 0.5% (w / w), at least 1% (w / w), at least 2.5% (w / w), at least 5% (w / w), at least 10% (w / w), or at least 15% (w / w) of a cryoprotectant, preferably a carbohydrate component, such as a sugar. In some embodiments, the liquid provided in step a) of the present disclosure comprises a cryoprotectant, optionally a carbohydrate, and optionally a sugar, in a concentration ranging from 0.1% to 40% (w / w), optionally in a concentration ranging from 1% to 20% (w / w), optionally in a concentration of 7.5%% (w / w). In certain embodiments, the liquid provided in step a) of the present disclosure comprises RNA at a concentration of about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, or about 95 μg / mL.

[0228] In vitro transcription of RNA

[0229] In some embodiments, RNA of the present disclosure includes RNA polynucleotides, such as messenger RNA (mRNA) or self-replicating RNA. mRNA is transcribed in vitro, for example, from a DNA template, referred to as an "in vitro transcription template."

[0230] In vitro transcription of RNA is well known in the art and described, for example, in International Publication No. WO2014 / 152027, which is incorporated herein by reference in its entirety. For example, in some embodiments, RNA transcripts are generated using a non-amplified linearized DNA template in an in vitro transcription reaction that produces the RNA transcript. In some embodiments, the RNA transcript is capped by enzymatic capping. In some embodiments, the RNA transcript is purified by chromatographic methods, for example, using an oligo-dT substrate. In some embodiments, the use of DNase is excluded. In some embodiments, the RNA transcript is synthesized from a non-amplified linear DNA template encoding a gene of interest by an enzymatic in vitro transcription reaction that utilizes T7 phage RNA polymerase and nucleotide triphosphates of the desired chemistry. Any number of RNA polymerases or variants can be used in the methods of the present disclosure. The polymerase can be selected from, but is not limited to, a phage RNA polymerase, e.g., T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or a mutant polymerase, such as a polymerase capable of incorporating modified nucleic acids and / or nucleotides, including, but not limited to, chemically modified nucleic acids and / or nucleotides.

[0231] As used herein, the term "terminus" or "terminus" when referring to a polypeptide or polynucleotide refers to the end of the polypeptide or polynucleotide, respectively. Such ends are not limited to only the first or last site of the polypeptide or polynucleotide, but can include additional amino acids or nucleotides in the terminal region. Polypeptide-based molecules can be characterized as having both an N-terminus (ending with an amino acid having a free amino group (NH2)) and a C-terminus (ending with an amino acid having a free carboxyl group (COOH)). Proteins are sometimes made of multiple polypeptide chains (multimers, oligomers) that are brought about by disulfide bonds or by non-covalent forces. These proteins have multiple N-terminus and C-terminus. Alternatively, the ends of the polypeptides can be modified to start or end at non-polypeptide sites, such as organic conjugates, as the case may be.

[0232] In some embodiments, the in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, encodes a 3' UTR, and a poly A tail. The particular nucleic acid sequence composition and length of the in vitro transcription template depends on the mRNA encoded by the template.

[0233] In some embodiments, the RNA described herein comprises an extended poly-A tail. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 35 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 40 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 45 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 50 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 55 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 60 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 65 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 70 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 80 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 90 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 95 and 100 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 95 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 90 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 85 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 80 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 75 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 70 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 65 nucleotides in length.In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 60 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 55 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 50 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 30 and 45 nucleotides in length. In some embodiments, the RNA described herein comprises a poly-A tail between 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or more than 140 nucleotides in length.

[0234] By "5' untranslated region" (5'UTR) is meant the region of an RNA (e.g., an mRNA or other coding RNA) located directly upstream (i.e., 5') from the start codon that does not encode a polypeptide (i.e., the first codon of an RNA, e.g., an mRNA or other coding RNA, a transcript translated by a ribosome).

[0235] "3' untranslated region" (3'UTR) refers to the region of an RNA (e.g., an mRNA or other coding RNA) that does not encode a polypeptide, located directly downstream (i.e., 3') from a stop codon (i.e., a codon in an RNA, e.g., an mRNA or other coding RNA, transcript that signals the end of translation).

[0236] In some embodiments, the RNA described herein has an extended 3'UTR. In some embodiments, the RNA described herein has a 3'UTR between 100 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 125 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 150 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 175 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 200 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 225 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 250 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 275 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 300 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 325 and 500 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 476 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 450 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 425 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 400 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 375 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 100 and 350 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR between 300 and 350 nucleotides in length. In some embodiments, the RNA described herein has a 3'UTR about 330 nucleotides in length.In some embodiments, the RNA described herein has a 3'UTR of about 270 nucleotides in length. In some embodiments, the RNA described herein comprises a 3'UTR of more than 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 440 nucleotides in length.

[0237] An "open reading frame" is a contiguous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA), and typically encodes a polypeptide (e.g., a protein). The sequence may further include additional elements, such as 5' and 3' UTRs, although it will be understood that these elements, unlike the ORF, need not necessarily be present in the disclosed vaccines or therapeutics.

[0238] A "poly-A tail" is a region of an RNA (e.g., an mRNA or other coding RNA) downstream, e.g., directly downstream (i.e., 3'), of a 3'UTR that contains multiple consecutive adenosine monophosphates. A poly-A tail can contain between 10 and 300 adenosine monophosphates. For example, a poly-A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, a poly-A tail contains between 50 and 250 adenosine monophosphates. In relevant biological settings (e.g., within a cell, in vivo), the poly(A) tail functions, for example, to protect the RNA from enzymatic degradation in the cytoplasm, and assists in transcription termination and / or export of the RNA from the nucleus and translation.

[0239] Polynucleotides

[0240] In some embodiments, the polynucleotides of the present disclosure function as messenger RNA (mRNA). By "messenger RNA" (mRNA) is meant any polynucleotide that encodes (at least one) polypeptide (natural, non-natural, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo. One of skill in the art will understand that, unless otherwise noted, the polynucleotide sequences recite "T" in exemplary DNA sequences, but when the sequence represents RNA (e.g., mRNA), "T" is replaced by "U".

[0241] Thus, any RNA polynucleotide encoded by a DNA identified by a particular sequence identification number can also include the corresponding RNA (e.g., mRNA or other coding RNA) sequence encoded by that DNA, where each "T" in the DNA sequence is replaced with a "U."

[0242] It should be understood that the RNA polynucleotides provided herein are synthetic molecules, i.e., they are not naturally occurring molecules. That is, the RNA polynucleotides of the present disclosure are isolated RNA polynucleotides. As known in the art, an "isolated polynucleotide" refers to a polynucleotide that is substantially physically isolated from other cellular materials (e.g., cells and / or systems that produce the polynucleotide) or from other materials that would prevent their use in the vaccines or therapeutics of the present disclosure. An isolated polynucleotide is substantially pure in that it is substantially isolated from substances that may be associated with a living or viral system. Thus, the RNA polynucleotide is not associated with a living or viral system, e.g., a cell or a virus. The RNA polynucleotide does not include viral components (e.g., viral capsids, viral enzymes, or other viral proteins, e.g., those necessary for viral-based replication), and the RNA polynucleotide is not packaged, enclosed, associated with, or otherwise associated with a virus or viral particle. In some embodiments, the RNA comprises a lipid nanoparticle that comprises, consists of, or consists essentially of an RNA polynucleotide (eg, an RNA polynucleotide encoding a VZV antigen).

[0243] Any sequence can be codon-optimized. Codon optimization methods are known in the art. In some embodiments, codon optimization can be used to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase RNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene constructs or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein, add, remove, shuffle protein domains, insert or delete restriction enzyme cleavage sites, modify ribosome binding and RNA degradation sites, adjust translation rates to allow various domains of a protein to fold properly, or reduce or remove problematic secondary structures in a polynucleotide. In some embodiments, open reading frame (ORF) sequences are optimized using optimization algorithms. In some embodiments, sequences encoding antigens, such as the spike protein of SARS-CoV-2, are codon-optimized. In some embodiments, the sequence encoding an antigen, eg, a VZV antigen, is codon optimized.

[0244] In some embodiments, the RNA can include at least one RNA polynucleotide encoding at least one antigenic polypeptide having at least one of the following modifications, at least one 5'-end cap, and formulation with lipid nanoparticles. 5'-capping of polynucleotides can be completed simultaneously during an in vitro transcription reaction using the following RNA cap analogs: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G according to the manufacturer's protocol to generate a 5' guanosine cap structure (New England BioLabs, Ipswich, Mass.). 5'-capping of modified RNA can be completed post-transcription using vaccinia virus capping enzyme to generate the "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, Mass.). Cap 1 structures can be generated using both vaccinia virus capping enzyme and a 2'-O-methyl-transferase to generate m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structures can be generated from Cap 1 structures by 2'-O-methylating the 5' penultimate nucleotide with a 2'-O-methyltransferase. Cap 3 structures can be generated from Cap 2 structures by 2'-O-methylating the 5' penultimate nucleotide with a 2'-O-methyltransferase. The enzymes can be from recombinant sources.

[0245] In some embodiments, the RNA can include a Cap1 cap. When transfected into mammalian cells, the modified RNA typically has a stability of 12 to 18 hours or more than 18 hours, such as 24, 36, 48, 60, 72, or more than 72 hours.

[0246] The Varicella-Zoster Virus (VZV) vaccines provided herein comprise at least one self-replicating ribonucleic acid (srRNA) polynucleotide encoding at least one VZV antigenic polypeptide.

[0247] In some embodiments, at least one RNA polynucleotide of the VZV vaccine is encoded by SEQ ID NO:4. In some embodiments, the srRNA sequence comprises the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the srRNA sequence comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 4.

[0248] In some embodiments, the RNA sequence comprises a nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the RNA sequence comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0249] Varicella-zoster virus (VZV)

[0250] VZV is an alpha-herpesvirus that exists as a spherical multi-layered structure with a diameter of approximately 200 nm. The viral genome is surrounded by a protein capsid structure, which is covered by an amorphous layer of tegument protein. These two structures are surrounded by a lipid envelope that encodes a 100 nm nucleocapsid, which is displayed on the outside of the virion and consists of 162 hexameric and pentameric capsomeres arranged in the form of an icosahedron, interspersed with viral glycoproteins, each about 8 nm in length. The tegument, which is composed of virally encoded proteins and enzymes, is located in the space between the nucleocapsid and the viral envelope. The viral envelope is obtained from the host cell membrane and contains virally encoded glycoproteins.

[0251] VZV is closely related to herpes simplex virus (HSV) and shares the majority of its genome homology. The VZV genome is the smallest human herpesvirus and encodes at least 71 unique proteins (ORF0 to ORF68) with three additional open reading frames (ORF69 to 71) that duplicate the previous open reading frames (ORF64 to 62, respectively). Only a portion of the encoded proteins form the structure of the virus particle. Among them are nine glycoproteins: ORF(gK), ORF9A(gN), ORF14(gC), ORF31(gB), ORF37(gH), ORF50(gM), ORF60(gL), ORF67(gI), and ORF68(gE). The known envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL, gN, and gM) correspond to those in HSV; however, there is no equivalent of HSV gD. VZV also cannot produce LAT (latency-associated transcript), which plays an important role in the establishment of latency in HSV (herpes simplex virus). The encoded glycoproteins gE, gI, gB, gH, gK, gL, gC, gN, and gM function in different steps of the viral replication cycle. The most abundant glycoprotein found in infected cells as well as in mature virions is glycoprotein E (gE, ORF68), which is the major component of the virion envelope and is essential for viral replication. Glycoprotein I (gI, ORG67) forms a complex with gE in infected cells, which promotes endocytosis of both glycoproteins and targets them to the trans-Golgi network (TGN) where the final viral envelope is obtained. Glycoprotein I (gI) is obtained within the TGN for VZV packaging and for efficient membrane fusion during VZV replication. VZV gE and gI are found complexed to each other on the infected host cell surface. Antibodies against gE, gB, and gH are abundant after natural infection and after vaccination and have been shown to neutralize viral activity in vitro.

[0252] antigen

[0253] The lyophilized RNA herein is designed to contain or code for a substance that generates an immune response in a subject. In addition to mRNA, the RNA described herein can be one of several non-coding types of RNA, such as ribosomal RNA (rRNA) or transfer RNA (tRNA). The term "RNA" or "RNA molecule" also encompasses other coding RNA molecules, such as viral RNA, retroviral RNA, self-replicating RNA (replicon RNA), small interfering RNA (siRNA), microRNA, small nuclear RNA (snRNA), small hairpin (sh) RNA, riboswitch, ribozyme, or aptamer.

[0254] In certain embodiments, the RNA is a long-chain RNA or a long-chain RNA molecule. The term "long-chain RNA" as used herein typically refers to an RNA molecule as described herein, preferably comprising at least 30 nucleotides. Alternatively, the long-chain RNA may comprise at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, or at least 500 nucleotides. The long-chain RNA may comprise at least 1000 nucleotides or at least 2000 nucleotides. The long-chain RNA, in the context of the present disclosure, may comprise 30 to 50,000 nucleotides, 30 to 20,000 nucleotides, 100 to 20,000 nucleotides, 200 to 20,000 nucleotides, 200 to 15,000 nucleotides, or 500 to 20,000 nucleotides. As used herein, the term "long RNA" is not limited to a particular type of RNA, but simply refers to the number of nucleotides contained in the RNA. In certain embodiments, the RNA used herein is a long mRNA.

[0255] In the present disclosure, the RNA can be a coding RNA molecule, encoding a protein or peptide, for example but not limited to, from a therapeutically active protein or peptide selected from adjuvant proteins, from an antigen, for example a tumor antigen, a pathogenic antigen (e.g. animal antigen, viral antigen, protozoan antigen, bacterial antigen), an allergen antigen, an autoimmune antigen or preferably a further antigen as defined herein, from an allergen, from an antibody, from an immunostimulatory protein or peptide, from an antigen-specific T-cell receptor or from any other protein or peptide suitable for a particular (therapeutic) application, and the coding RNA molecule can be delivered to a cell, tissue or organism, and the protein can be subsequently expressed in said cell, tissue or organism. In a particular embodiment, the RNA provided in the liquid of step a) of the present disclosure is an RNA molecule, and "step a) of the present disclosure" refers to step a) of the method of freeze-drying an RNA composition, in which a liquid comprising RNA and at least one suitable protective agent is provided.

[0256] In certain embodiments, the RNA in the liquid provided in step a) of the present disclosure can be an immunostimulatory RNA molecule, such as any RNA molecule known in the art that is capable of eliciting an immune response, preferably an innate immune response. Such an immunostimulatory RNA can be any (double-stranded or single-stranded) RNA, for example a coding RNA, as defined herein. In certain embodiments, the immunostimulatory RNA is a non-coding RNA. The immunostimulatory RNA can be a single-stranded, double-stranded or partially double-stranded RNA, optionally a single-stranded RNA, or a circular or linear RNA, preferably a linear RNA. In various embodiments, the immunostimulatory RNA can be a linear single-stranded RNA. Even more preferably, the immunostimulatory RNA can be a long linear single-stranded RNA.

[0257] Immunostimulatory RNA can also occur as short RNA oligonucleotides. As used herein, immunostimulatory RNA can be selected from any class of RNA molecule found in nature or prepared synthetically and capable of inducing an innate immune response and supporting an antigen-induced adaptive immune response.

[0258] In some embodiments, the antigenic polypeptide is a VZV glycoprotein. For example, the VZV glycoprotein can be VZV gE, gI, gB, gH, gK, gL, gC, gN, or gM. The UniProtKB accession numbers for the glycoproteins are gI: P09258; gB: P09257; gH: P09260; gK: P09261; gL: Q71S15 and Q775I7; gC: P09256; gN: Q0Q872; and gM: P09298. In some embodiments, the antigenic polypeptide is a VZV gE polypeptide. The antigenic polypeptide can be encoded by one or more nucleotide sequences. Such nucleotide sequences encoding the antigenic polypeptide can be included in a vector, such as srRNA.

[0259] The present disclosure includes variant VZV antigen polypeptides. In some embodiments, the variant VZV antigen polypeptide is a variant VZV gE polypeptide. The variant VZV gE polypeptide is designed to avoid ER / Golgi retention of the polypeptide, resulting in increased surface expression of the antigen. In some embodiments, the variant gE polypeptide is truncated to remove the ER retention portion or the cytoplasmic tail portion of the polypeptide. In some embodiments, the variant VZV gE polypeptide is mutated to reduce ER / Golgi / TGN localization of the VZV polypeptide. Such modifications inhibit ER trapping, thereby facilitating trafficking to the cell membrane.

[0260] Thus, in some embodiments, the VZV glycoprotein is a variant gE polypeptide. VZV gE has a targeting sequence for the TGN at the C-terminus and is transported from the ER to the TGN in infected and gE-transfected cells. Most gE in the TGN appears to be retrieved by endocytosis from the plasma membrane and delivered to the TGN by endosomes, followed by recycling to the plasma membrane. gE accumulates in the TGN along with other VZV proteins (e.g., coat proteins) that are associated with the production of fully encapsulated VZV virions. Thus, mutations that reduce TGN localization and endocytosis aid in the transport of gE to the plasma membrane.

[0261] A variant VZV gE polypeptide can be any truncated VZV gE polypeptide that lacks the anchor domain (ER retention domain). For example, a variant VZV gE polypeptide can be a truncated VZV gE polypeptide that includes at least amino acids 1-573 of the sequence set forth in SEQ ID NO:3, as well as polypeptide fragments having fragment sizes within the recited size ranges. In one embodiment, a truncated VZV gE polypeptide includes amino acids 1-573 of the sequence set forth in SEQ ID NO:3. In some embodiments, a variant VZV gE polypeptide is a truncated polypeptide that lacks the carboxy-terminal tail domain. Thus, in some embodiments, a truncated VZV gE polypeptide includes amino acids 1-573 of the sequence set forth in SEQ ID NO:3.

[0262] In some embodiments, the VZV gE comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 75% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 97% sequence identity to the amino acid sequence of SEQ ID NO:3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the VZV gE comprises an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0263] In some embodiments, the VZV gE comprises the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, the VZV gE comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 1.

[0264] In some embodiments, the variant VZV gE polypeptide has at least one mutation in one or more motifs associated with ER retention, wherein the mutation in the one or more motifs results in reduced retention of the VZV gE polypeptide in the ER and / or Golgi. For example, the variant VZV gE polypeptide can be a full-length or truncated VZV gE polypeptide having a Y569A mutation.

[0265] In some embodiments, the RNA induces an immune response against a coronavirus selected from the group consisting of 229E (alpha coronavirus), NL63 (alpha coronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), MERS-CoV (MERS), SARS-CoV (SARS), and SARS-CoV-2 (COVID-19). In some embodiments, the virus is SARS-CoV-2. In some embodiments, the RNA induces an immune response against SARS-CoV-2.

[0266] In some embodiments, the RNA induces an immune response against a SARS-CoV-2 variant selected from the group consisting of Wuhan-Hu-1, alpha, beta, gamma, delta, epsilon, eta, iota, kappa, 1.617.3, mu, zeta, and o. In some embodiments, the RNA induces an immune response against a SARS-CoV-2 variant selected from the group consisting of Wuhan-Hu-1 and Delta.

[0267] In some embodiments, the antigenic polypeptide comprises a viral envelope protein, a viral spike protein, a viral membrane protein, or a viral capsid protein. In some embodiments, the antigenic polypeptide comprises a viral spike protein.

[0268] self-replicating RNA

[0269] Polynucleotides can also include RNA, such as self-replicating RNA. The RNA described herein can be a self-replicating RNA. A self-replicating RNA molecule (replicon), even in the absence of any proteins, can result in the production of multiple daughter RNAs by transcription from itself (by antisense copies generated from itself) when delivered to a vertebrate cell. Thus, a self-replicating RNA molecule can typically be a ±strand molecule that can be directly translated after delivery to a cell, and this translation results in an RNA-dependent RNA polymerase that then produces both antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA can result in the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, can be translated into themselves, resulting in the in situ expression of the encoded immunogen, or they can be transcribed to produce additional transcripts with the same sense as the delivered RNA, resulting in the in situ expression of the immunogen. The overall result of this series of transcriptions is a large amplification in the number of introduced replicon RNAs, with the encoded immunogen becoming the predominant polypeptide product of the cell.

[0270] One suitable system to achieve self-replication in this way is to use alphavirus-based replicons. These replicons can be ± strand RNA, which after delivery to the cell results in the translation of a replicase (or replicase-transcriptase). The replicase can be translated as a polyprotein, the self-cleavage of which results in a replication complex that makes copies of the genome - / - strand of the ± strand delivered RNA. These - / - strand transcripts can transcribe themselves to obtain further copies of the ± strand parent RNA, or can result in subgenomic transcripts that code for immunogens. Translation of the subgenomic transcripts can thus result in in situ expression of the immunogen by the infected cell. Suitable alphavirus replicons can be Sindbis virus, Semliki Forest virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, and the like. Mutant or wild-type virus sequences can be used, for example, the attenuated TC83 mutant of VEEV has been used in the replicons.

[0271] The self-replicating RNA molecule can encode (i) an RNA-dependent RNA polymerase capable of transcribing RNA from the self-replicating RNA molecule, and (ii) an immunogen. The polymerase can be an alphavirus replicase, including, for example, one or more of the alphavirus proteins nsp1, nsp2, nsp3, and nsp4. While naturally occurring alphavirus genomes encode structural virion proteins in addition to nonstructural replicase polyproteins, the self-replicating RNA molecules described herein may lack one or more, or all, of the alphavirus structural proteins. Thus, the self-replicating RNA can result in the production of its own genomic RNA copies within the cell, rather than the production of RNA-containing virions. The inability to produce these virions means that, unlike wild-type alphaviruses, the self-replicating RNA molecules generally do not perpetuate themselves in an infectious form. The alphavirus structural proteins used for perpetuation in wild-type viruses are typically absent in the self-replicating RNA molecules described herein, and in their place are placed genes encoding the immunogen of interest, with the subgenomic transcripts encoding the immunogen rather than the structural alphavirus virion proteins.

[0272] Thus, a self-replicating RNA molecule can have two open reading frames: the first (5') open reading frame encodes a replicase and the second (3') open reading frame encodes an immunogen. In some embodiments, the RNA can have an additional (e.g., downstream) open reading frame, e.g., encoding a further immunogen (see below) or encoding an accessory polypeptide.

[0273] The self-replicating RNA molecule can have a variety of lengths, but is typically 5,000 to 25,000 nucleotides long, for example, 8,000 to 15,000 nucleotides, or 9,000 to 12,000 nucleotides long. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the self-replicating RNA comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 2.

[0274] In some embodiments, described herein are self-replicating RNA molecules that include one or more components (e.g., Cap1, modified 3'UTR, extended polyA tail, or combinations thereof) that improve gene of interest (GOI) expression, immunogenicity, scalability, and manufacturability, or combinations thereof. In some embodiments, the self-replicating RNA molecules include at least 1.5X, 2.0X, 2.5X, 3.0X, or more GOI expression compared to unmodified vectors. In some embodiments, the srRNA vectors include at least 2X, 5X, 10X, 20X, 25X, 30X, or more improved immune response compared to unmodified vectors. In some embodiments, the unmodified srRNA vectors include a polyA tail of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, or 140 nucleotides in length. In some embodiments, an unmodified srRNA vector comprises a cap that is not Cap 1 (e.g., Cap 0). In some embodiments, an unmodified srRNA vector does not comprise a modification in one or more components (the cap, UTR, or polyA tail) compared to the parent vector.

[0275] The self-replicating RNA molecules described herein can have a 5' cap (e.g., 7-methylguanosine). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of the RNA molecules useful in the present disclosure can have a 5' triphosphate group. In capped RNA, this can be linked to the 7-methylguanosine via a 5'-5' bridge. In some embodiments, the RNA cap can include m7G (cap 0), m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated) (cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or an anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated).

[0276] The self-replicating RNA molecule can have a 3' poly A tail. The self-replicating RNA molecule can also include a poly A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.

[0277] In some embodiments, the self-replicating RNA described herein comprises an extended poly-A tail. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 35 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 40 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 45 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 50 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 55 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 60 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 65 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 70 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 80 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 90 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 95 and 100 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 95 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 90 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 85 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 80 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 75 nucleotides in length.In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 70 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 65 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 60 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 55 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 50 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 30 and 45 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a poly-A tail between 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or more than 140 nucleotides in length.

[0278] In some embodiments, the self-replicating RNA described herein has an extended 3'UTR. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 180-400 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 100-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 125-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 150-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 175-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 200-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 225-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR that is 250-500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 275 and 500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 300 and 500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 325 and 500 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 476 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 450 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 425 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 400 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 375 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR between 100 and 350 nucleotides in length.In some embodiments, the self-replicating RNA described herein has a 3'UTR of 300-350 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR of about 330 nucleotides in length. In some embodiments, the self-replicating RNA described herein has a 3'UTR of about 270 nucleotides in length. In some embodiments, the self-replicating RNA described herein comprises a 3'UTR of more than 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 440 nucleotides in length.

[0279] In some embodiments, the 3'UTR comprises a nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 70% (e.g., 75%, 80%, 90%, 95%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 75% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the 3'UTR comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0280] In some embodiments, the self-replicating RNA molecules described herein include a cap (e.g., m7G (cap 0), m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated) (cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), or anti-reverse cap analog (ARCA), optionally m7G or m7GpppNm- (where Nm represents any nucleotide that is 2'O methylated)), a 5'UTR, one or more alphavirus proteins (e.g., nsp1, nsp2, nsp3, and nsp4), a gene of interest (e.g., VZV), a 3'UTR, and a polyA tail. In some embodiments, the 3'UTR is an extended UTR having a length of more than about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, or 440 nucleotides. In some embodiments, the 3'UTR has a length of about 330 nucleotides. In some embodiments, the 3'UTR has a length of about 270 nucleotides. In some embodiments, the polyA tail has a length of about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or more than 140 nucleotides. In some embodiments, the polyA tail has a length of about 65 nucleotides. In some embodiments, the polyA tail has a length of about 95 nucleotides.

[0281] In some embodiments, a self-replicating RNA molecule or vector described herein comprises a cap, a 5'UTR, one or more alphavirus proteins (e.g., nsp1, nsp2, nsp3, and nsp4), a gene of interest (e.g., VZV), a 3'UTR having about 180 to 400 (e.g., about 270 or about 330 nucleotides) nucleotides in length, and a polyA tail having about 40 to about 100 nucleotides in length (e.g., about 40, about 65, or about 95 nucleotides in length). In some embodiments, the self-replicating RNA molecules described herein include cap1, a 5'UTR, one or more alphavirus proteins (e.g., nsp1, nsp2, nsp3, and nsp4), a gene of interest (e.g., VZV), a 3'UTR having a length of about 100 to about 400 (e.g., about 270 or about 330 nucleotides), and a polyA tail having a length of about 40 to about 100 nucleotides (e.g., about 40, about 65, or about 95 nucleotides). In some embodiments, a self-replicating RNA (srRNA) molecule or vector comprises, in 5' to 3' order: a) Cap1 cap, b) 5'UTR, c) one or more structural proteins; d) gene of interest (GOI); e) 3'UTR having about 180 to about 400 nucleotides; and f) polyA tail having about 60 to about 100 nucleotides. In some embodiments, the srRNA molecule or vector comprises at least 1.5X, 2.0X, 2.5X, 3.0X, or more GOI expression compared to an unmodified molecule or vector. In some embodiments, the srRNA molecule or vector comprises at least 2X, 5X, 10X, 20X, 25X, 30X, or more improved immune response compared to an unmodified molecule or vector. In some embodiments, the GOI is a Varicella-Zoster Virus (VZV) antigen. In some embodiments, the VZV antigen comprises a VZV glycoprotein E (gE) antigen.

[0282] Self-replicating RNA molecules are typically single-stranded. Single-stranded RNA can generally initiate adjuvant effects by binding to TLR7, TLR8, RNA helicase, and / or PKR. RNA delivered in double-stranded form (dsRNA) can bind to TLR3, a receptor that can also be triggered by dsRNA, either during replication of the single-stranded RNA or in the secondary structure of the single-stranded RNA.

[0283] The self-replicating RNA molecules described herein can be prepared by in vitro transcription (IVT). IVT can use cDNA templates that are produced and propagated in bacteria in plasmid form or that are produced synthetically (e.g., by gene synthesis and / or polymerase chain reaction (PCR) recombinant methods). For example, a DNA-dependent RNA polymerase (such as bacteriophage T7, T3, or SP6 RNA polymerase) can be used to transcribe the RNA from a DNA template. Appropriate capping and polyA addition reactions can be used as needed (although the polyA of the replicon is usually encoded within the DNA template). These RNA polymerases can have stringent requirements for the transcribed 5' nucleotides, and in some embodiments, these requirements must be consistent with those of the encoded replicase to ensure that the IVT-transcribed RNA can function efficiently as a substrate for the self-encoded replicase.

[0284] immunogen

[0285] The RNA molecules described herein can encode a polypeptide immunogen. The self-replicating RNA molecules described herein can encode a polypeptide immunogen. After administration of the RNA, the immunogen can be translated in vivo and elicit an immune response in the recipient. The immunogen can elicit an immune response against an antigen, a virus, and / or a viral antigen. The immune response can include an antibody response. The immune response can include B cells, CD4+ T cells, and / or CD8+ T cells. The immune response can include CD8+ T cells. The immunogen typically elicits an immune response that recognizes a corresponding antigen, such as a viral polypeptide. The immunogen is typically a surface polypeptide, such as an adhesin, hemagglutinin, envelope glycoprotein, spike glycoprotein, etc. In some embodiments, the immunogen elicits an immune response against the spike glycoprotein of SARS-CoV-2. In some embodiments, the immunogen elicits an immune response against the Varicella-Zoster virus. In some embodiments, the immunogen is VZV glycoprotein E (gE).

[0286] In some embodiments, the VZV srRNA vaccines described herein provide an improved immune response, ie, the immune response is improved by about 1X, 2X, 3X, 4X, 5X, 6X, or more, compared to a subject who does not receive the srRNA vaccine or who receives a different VZV vaccine.

[0287] In some embodiments, the VZV srRNA vaccines described herein provide an improved humoral response, ie, the humoral response is improved by about 50X, 60X, 70X, 80X, 90X, 100X, 110X, 120X, 130X, 140X, 150X, 160X, 170X, 180X, 190X, 200X, or more, compared to a subject that does not receive the srRNA vaccine or receives a different VZV vaccine.

[0288] Nanoparticle formulations

[0289] The present disclosure provides a method for mixing lyophilized RNA with a delivery vehicle. The delivery vehicle can be a non-virion, i.e., a particle that is not a virion. Thus, in some embodiments, the delivery vehicle does not include a protein capsid. By eliminating the need to create a capsid, the delivery vehicle does not utilize a packaging cell line, which makes it easier to scale up for commercial manufacturing and minimizes the risk of accidentally producing dangerous infectious viruses. Various materials are suitable delivery cells that can deliver RNA to vertebrate cells in vivo. Two types of delivery materials are (i) amphiphilic lipids that can form liposomes, and (ii) non-toxic and biodegradable polymers that can form microparticles. Other delivery methods include, but are not limited to, exosomes and cationic nanoemulsions.

[0290] When delivered by liposomes, RNA can be encapsulated or adsorbed, and when delivered by polymeric microparticles, RNA can be encapsulated or adsorbed. The third delivery material is a particulate reaction product of a polymer, a crosslinker, RNA, and a charged monomer. In certain embodiments, the delivery particles described herein comprise liposomes that adsorb RNA molecules encoding antigens.

[0291] RNA can be encapsulated in liposomes. This means that the RNA inside the particle is separated from any external medium by the delivery material, and it has been found that the encapsulation protects the RNA from RNase degradation. The encapsulation can take a variety of forms. For example, in some embodiments, the delivery material forms an outer layer around an aqueous RNA-containing core. In some embodiments, the RNA can be adsorbed to the particle. In some embodiments, this means that unlike the RNA genome of natural viruses, the RNA is not separated from any external medium by the delivery material. In some embodiments, the RNA is formulated in lipid nanoparticles.

[0292] In some embodiments, the RNA delivery vehicle is a nanoparticle (e.g., LNP) comprising at least one lipid. In some embodiments, the lipid comprises an ionizable lipid of formula I. [ka] R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl R4 and R5 are each independently C1-C 10 is alkyl; R6 and R7 are each independently C1-C 10 Alkyl, C1-C 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; R8 and R9 are each independently C1-C 30 It is an alkyl.

[0293] In some embodiments, L is [ka] or [ka] It is.

[0294] In some embodiments, the lipid is an ionizable lipid of formula II [ka] Including, During the ceremony, R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18 is an alkyl group Q1 and Q2 are each independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-;-SS-; and R6 and R7 are each independently C1-C 32 It is an alkyl.

[0295] In some embodiments, R1 and R2 are methyl.

[0296] In some embodiments, Q1 and Q2 are each independently -C(O)-O- or -OC(O)-.

[0297] In some embodiments, R3 is a straight chain C3 alkyl.

[0298] In some embodiments, R4 and R5 are each independently a C5-C10 alkyl group.

[0299] In some embodiments, R and R are each independently C-C alkyl. In some embodiments, R and R are each independently C-C alkyl.

[0300] In some embodiments, the lipid is [ka] It can be.

[0301] In some embodiments, the lipid is [ka] It can be.

[0302] In some embodiments, the lipid is [ka] It can be.

[0303] In some embodiments, the lipid is [ka] It can be.

[0304] In some embodiments, the lipid is [ka] It can be.

[0305] In some embodiments, the lipid is [ka] It can be.

[0306] In some embodiments, the lipid is [ka] It can be.

[0307] In some embodiments, the lipid is [ka] It can be.

[0308] In some embodiments, pharma- ceutically acceptable salts of the lipids described herein include those derived from pharma- ceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, trifluoroacetate, and undecanoate.

[0309] In some embodiments, the lipid can be a cationic lipid, also called an ionizable lipid. In some embodiments, useful cationic lipids generally contain a nitrogen atom that is positively charged under physiological conditions, for example, as a tertiary or quaternary amine. This nitrogen can be present in the hydrophilic head group of an amphiphilic surfactant. The lipid can be selected from, but is not limited to, 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP), 3'-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC cholesterol), dimethyldioctadecyl-ammonium (DDA, e.g., bromide), 1,2-dimyristoyl-3-trimethyl-ammonium propane (DMTAP), dipalmitoyl (C16:0) trimethylammonium propane (DPTAP), distearoyltrimethylammonium propane (DSTAP). Other useful cationic lipids are benzalkonium chloride (BAK), benzethonium chloride, cetramid (containing tetradecyltrimethylammonium bromide and, optionally, small amounts of dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide), cetylpyridinium chloride (CPC), cetyltrimethylammonium chloride (CTAC), N,N',N'-polyoxyethylene(10)-N-tallow-1,3-diaminopropane, dodecyltrimethylammonium bromide, hexadecyltrimethyl-ammonium bromide, mixed alkyl-trimethyl-ammonium bromide, benzyldimethyldodecylammonium chloride, and tetradecyltrimethylammonium bromide. , benzyl dimethyl hexadecyl-ammonium chloride, benzyl trimethyl ammonium methoxide, cetyl dimethyl ethyl ammonium bromide, dimethyl dioctadecyl ammonium bromide (DDAB), methyl benzethonium chloride, decamethonium chloride, methyl mixed trialkyl ammonium chloride, methyl trioctyl ammonium chloride, N,N-dimethyl-N-[2(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]-ethoxy)ethyl]-benzenemethanaminium chloride (DEBDA), dialkyl dimethyl ammonium salt, [1-(2,3-dioleyloxy)-propyl]-N,N,N,Trimethylammonium chloride, 1,2-diacyl-3-(trimethylammonio)propane (acyl groups = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-diacyl-3-(dimethylammonio)propane (acyl groups = dimyristoyl, dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester, cholesteryl (4'-trimethylammonio)butanoate, N-alkylpyridinium salts (e.g., cetylpyridinium bromide and cetylpyridinium chloride), diphenyl phosphate), N-alkyl piperidinium salts, dicationic bolaform electrolytes (Cl2Me6;Cl2BU6), dialkyl glyceryl phosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine, cholesterol hemisuccinic acid choline ester, dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanol-amidospermine (DPPES), lipopoly-L(or D)-lysine (LPLL, LPDL), poly-L(or D)-lysine conjugated to N-glutarylphosphatidylethanolamine, didodecyl glutamate esters with pendant amino groups (C, 12 GluPhC n N + ), ditetradecyl glutamate ester with pendant amino groups (C12GluPhC n N | ), cationic derivatives of cholesterol including, but not limited to, lipopolyamines, cholesteryl-3β-oxysuccinic acid amide ethylene trimethyl ammonium salt, cholesteryl-3β-oxysuccinic acid amide ethylene dimethylamine, cholesteryl-3β-carboxyamido ethylene trimethyl ammonium salt, and cholesteryl-3β-carboxyamido ethylene dimethylamine.

[0310] In some embodiments, the RNA delivery vehicle is a nanoparticle that comprises at least one lipid.In some embodiments, the lipid can be a neutral lipid.In some embodiments, the lipid can be a phospholipid. The phospholipids are DDPC, 1,2-didecanoyl-sn-glycero-3-phosphatidylcholine, DEPA, 1,2-dierucoyl-sn-glycero-3-phosphate, DEPC, 1,2-erucoyl-sn-glycero-3-phosphatidylcholine, DEPE, 1,2-dierucoyl-sn-glycero-3-phosphatidylethanolamine, DEPG, 1,2-dipalmitoylphosphatidylglycerol, DLOPC, 1,2-linoleoyl-sn-glycero-3-phosphatidylcholine, DLPA, 1,2-dilauroyl-sn-glycero-3-phosphate, DLPC, 1,2-dilauroyl-sn-glycero-3-phosphatidylcholine, DLPE, 1,2-dilauroyl-sn-glycero-3-phosphatidylethanolamine, DLPG 1,2-dilauroyl-sn-glycero-3 (phosphorylglycerol), DLPS 1,2-dilauroyl-sn-glycero-3-phosphatidylserine, DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPA, 1,2-dimyristoyl-sn-glycero-3-phosphate, DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine, DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphatidylethanolamine, DMPG, 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, DMPS, 1,2-dimyristoyl-sn-glycero-3-phosphatidylserine , DOPA, 1,2-dioleoyl-sn-glycero-3-phosphate, DOPC, 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine, DOPE, 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, DOPG, 1,2-dipalmitoylphosphatidylglycerol, DOPS, 1,2-dioleoyl-sn-glycero-3-phosphatidylserine, DPPA, 1,2-dipalmitoyl-sn-glycero-3-phosphate, DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, DPPE, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, DPPG, 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol, DPPS, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylserine, DPyPE, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, DSPA, 1,2-distearoyl-sn-glycero-3-phosphate, DSPC, 1,2-distearoyl-sn-glycero-3-phosphatidyl Dyllcholine, DSPE, 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine, DSPG, 1,2-distearoyl-sn-glycero-3-phosphorylglycerol, DSPS, 1,2-diundecanoyl-sn-glycero-phosphocholine, DUPC, 1,2-distearoyl-sn-glycero-3-phosphatidylserine, EPC, Egg-PC, HEPC, Hydrogenated Egg PC, HSPC, High Purity Hydrogenated Soy PC, HSPC, Hydrogenated Soy PC, Lysopc Myristic, 1-Myristoyl-sn-glycero-3-phosphatidylcholine, LYSOPC PALMITIC, 1-Palmitoyl-sn-glycero-3-phosphatidylcholine, LYSOPC STEARIC, 1-stearoyl-sn-glycero-3-phosphatidylcholine, bovine milk sphingomyelin, MPPC, 1-myristoyl,2-palmitoyl-sn-glycero-3-phosphatidylcholine, MSPC, 1-myristoyl,2-stearoyl-sn-glycero-3-phosphatidylcholine, PMPC, 1-palmitoyl,2-myristoyl-sn-glycero-3-phosphatidylcholine, POPC, 1-palmitoyl,2-oleoyl-sn-glycero-3-phosphatidylcholine, POPE, 1 -palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine, POPG, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol)], PSPC, 1-palmitoyl,2-stearoyl-sn-glycero-3-phosphatidylcholine, SMPC, 1-stearoyl,2-myristoyl-sn-glycero-3-phosphatidylcholine, SOPC, 1-stearoyl,2-oleoyl-sn-glycero-3-phosphatidylcholine, SPPC, 1-stearoyl,The phosphatidylcholine may be selected from, but is not limited to, 2-palmitoyl-sn-glycero-3-phosphatidylcholine.

[0311] In some embodiments, the RNA delivery vehicle is a nanoparticle comprising at least one lipid. In some embodiments, the lipid can be a PEGylated lipid (PEG). In some embodiments, the PEGylated lipid comprises a polyethylene glycol moiety. In some embodiments, the PEGylated lipid can include, but is not limited to, a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, or a combination thereof. In some embodiments, the PEGylated lipid can be a PEG-c-DOMG, a PEG-DMG, a PEG-DLPE, a PEG-DMPE, a PEG-DPPC, or a PEG-DSPE lipid. In some embodiments, the PEGylated lipid is a DMG-PEG, i.e., a PEG-conjugated 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]. In some embodiments, the lipid is DMG-PEG 2000, i.e., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), DMG-PEG 3500, DMG-PEG 5000, DTDAM-PEG 2000 (ALC-0159), DTDAM-PEG 5000, DMG-C-PEG 2000, DMG-C-PEG 5000, DSG-PEG 2000, DSG-PEG 5000, DPG-PEG 2000, DPG-PEG 5000, or any combination thereof.

[0312] In some embodiments, the RNA delivery vehicle is a nanoparticle that comprises at least one lipid. In some embodiments, the lipid can be a structured lipid. In some embodiments, the structured lipid can include, but is not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, or combinations thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof.

[0313] In some embodiments, the lipid nanoparticle (LNP) formulation comprises, consists essentially of, or consists of (i) neutral phospholipids, (ii) a sterol, e.g., cholesterol, (iii) optionally a PEGylated lipid, and (iv) an ionizable lipid, in molar ratios ranging from 5%-20% neutral phospholipids, 18.5%-58.5% sterol, 1%-4% PEGylated lipid, and 30%-70% ionizable lipid, with the sum of the lipid molar ratios being 100%, optionally 10:40.5:1.5:48.

[0314] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000:and ionized lipid, with molar ratios ranging from DSPC: 5%-20%, cholesterol: 18.5%-58.5%, DMG-PEG 2000: 1%-4%, ionized lipid: 30%-70%, and the sum of the molar ratios of lipids is 100%.

[0315] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionized lipid in molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 10.5%-43% DMG-PEG 2000, and ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0316] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000:ionized lipid with molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and 40%-50% ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0317] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000:ionized lipid with molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and 45%-50% ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0318] In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000:ionized lipid with molar ratios ranging from 5%-20% DSPC, 30%-55% cholesterol, 1%-4% DMG-PEG 2000, and 46%-49% ionized lipid, with the sum of the molar ratios of lipids being 100%.

[0319] In some embodiments, the ionizable lipid comprises about 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, or 49% of the LNP. In some embodiments, the LNP comprises DSPC:cholesterol:DMG-PEG 2000: and ionizable lipid in a molar ratio of 10:40.5:1.5:48.

[0320] In some embodiments, the pH of the LNP is 5-6.

[0321] In some embodiments, the RNA of the present disclosure can be formulated in lipid nanoparticles having a diameter of about 40 nm to 300 nm, optionally, the particle size of the LNP is less than 160 nm, and optionally, the particle size of the LNP is about 140 to 160 nm.

[0322] Lipids

[0323] As used herein, lipids of formula I: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; Q1, Q2, and Q3 are each independently -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; L is C1-C3 alkyl; R4 and R5 are each independently C1-C 10 is alkyl; R6 and R7 are each independently C1-C 10 Alkyl, or C1-C 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -C(O)O-, -OC(O)-, -SS-, -C(O)S-, -SC(O)-, -OC(S)-, or -C(S)O-; R8 and R9 are each independently C1-C 30 It is an alkyl.

[0324] In some embodiments, L is [ka] or [ka] It is.

[0325] As used herein, a lipid of formula II: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R1 and R2 are each independently C1-C6 alkyl; R3 is C1-C5 alkyl; R4 and R5 are each independently C1-C 18 is alkyl; Q1 and Q2 are each independently -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-; -SS-; R6 and R7 are each independently C 18 -C 32 It is an alkyl.

[0326] In some embodiments, R1 and R2 are methyl.

[0327] In some embodiments, Q1 and Q2 are each independently -C(O)-O- or -OC(O)-. In some embodiments, R3 is C3 alkyl.

[0328] In certain embodiments, the lipid is [ka] [ka] [ka] ; [ka] and [ka] is selected from the group consisting of:

[0329] In certain embodiments, provided herein is a pharma- ceutically acceptable composition comprising a lipid of any of the above embodiments and a pharma- ceutically acceptable carrier.

[0330] In some embodiments, pharma- ceutically acceptable salts of the lipids described herein include those derived from pharma- ceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, trifluoroacetate, and undecanoate.

[0331] Treatment method

[0332] Provided herein are compositions (eg, pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of diseases or conditions in humans and other mammals.

[0333] The disclosure herein provides a method for mixing lyophilized RNA with liquid LNP solution to make pharmaceutical composition.In certain embodiments, liquid LNP solution is added to lyophilized RNA.In certain embodiments, lyophilized RNA and liquid LNP solution are mixed at room temperature.In certain embodiments, lyophilized RNA and liquid LNP solution are mixed before clinical use.

[0334] Preventive protection from antigens can be achieved after administration of the disclosed RNA vaccine or therapeutic agent.In certain embodiments, administering the vaccine or therapeutic agent twice is sufficient.Although less desirable, the vaccine or therapeutic agent can be administered to infected individuals to achieve a therapeutic response.

[0335] A method of inducing an immune response in a subject against an antigen is provided in an aspect of the present disclosure. The method involves administering to the subject an RNA vaccine or therapeutic comprising an RNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide, thereby inducing an immune response in the subject. An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide.

[0336] As used herein, a "prophylactically effective dose" is a therapeutically effective dose that prevents infection by a virus at a clinically acceptable level. In some embodiments, a therapeutically effective dose is a dose listed on the package insert for a vaccine or therapeutic. As used herein, a conventional vaccine refers to a vaccine other than an RNA vaccine or therapeutic of the present disclosure. For example, a conventional vaccine includes, but is not limited to, a live microbial vaccine, an inactivated microbial vaccine, a subunit vaccine, a protein antigen vaccine, a DNA vaccine, a VLP vaccine, and the like. In an exemplary embodiment, a conventional vaccine is a vaccine that has received regulatory approval and / or is registered by a national drug regulatory agency, such as the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in the United States.

[0337] A method of inducing immune response in a subject is provided in the present disclosure.The method involves administering to a subject RNA comprising an RNA polynucleotide.In certain embodiments, the RNA polynucleotide has an open reading frame encoding at least one antigen polypeptide, thereby inducing a specific immune response against the antigen polypeptide in the subject, wherein the anti-antigen polypeptide antibody titer in the subject increases after vaccination.

[0338] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for the prevention and / or treatment of VZV in humans and other mammals. VZV RNA vaccines can be used as therapeutic or prophylactic agents. They can be used to prevent and / or treat infectious diseases. In exemplary embodiments, the VZV RNA vaccines disclosed herein are used to provide prophylactic protection against chickenpox and shingles. Chickenpox is an acute infectious disease caused by VZV. Primary varicella-zoster virus infection, which causes chickenpox (chicken pox), can lead to complications, including viral or secondary bacterial pneumonia. Even when the clinical symptoms of chickenpox resolve, VZV remains dormant in the nervous system of infected humans in the trigeminal and dorsal root ganglia and can reactivate later in life, migrating back from the sensory ganglia to the skin where it produces a disease (rash) known as herpes zoster or shingles, which can also cause a number of neurological pathologies ranging from aseptic meningitis to encephalitis. The VZV vaccine of the present disclosure can be used to prevent and / or treat both the primary infection (chickenpox) and also the reactivated viral infection (herpes zoster or shingles), and may be particularly useful in preventing and / or treating shingles in immunocompromised and elderly patients, particularly for the prevention and / or reduction of its severity and / or duration.

[0339] A method of inducing an immune response in a subject against an antigen (e.g., VZV) is provided in an embodiment of the present disclosure. The method involves administering to the subject an srRNA vaccine (e.g., VZV srRNA) comprising at least one srRNA polynucleotide having an open reading frame encoding at least one antigenic polypeptide (e.g., VZV antigen), thereby inducing an immune response in the subject specific to the antigenic polypeptide (e.g., VZV antigen). An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigenic polypeptide.

[0340] A method of inducing an immune response in a subject against an antigen (e.g., a VZV antigen) is provided in an aspect of the present disclosure. The method involves administering to the subject an RNA vaccine comprising at least one RNA polynucleotide having an open reading frame encoding at least one antigen polypeptide (e.g., a VZV antigen), thereby inducing a specific immune response in the subject against the antigen polypeptide (e.g., a VZV antigen), wherein the anti-antigen polypeptide antibody titer in the subject is increased after vaccination compared to the anti-antigen polypeptide antibody titer in the subject vaccinated with a prophylactically effective dose of a conventional vaccine against the antigen (e.g., a VZV antigen). An "anti-antigen polypeptide antibody" is a serum antibody that specifically binds to an antigen polypeptide.

[0341] A prophylactically effective dose is a therapeutically effective dose that prevents infection by the virus at a clinically acceptable level. In some embodiments, a therapeutically effective dose is a dose listed on the package insert for the vaccine. As used herein, a conventional vaccine refers to a vaccine other than the srRNA vaccine of the present disclosure. For example, conventional vaccines include, but are not limited to, live microbial vaccines, inactivated microbial vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines, and the like. In an exemplary embodiment, a conventional vaccine is a vaccine that has received regulatory approval and / or is registered by a national drug regulatory agency, such as the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA) in the United States.

[0342] Administration Method

[0343] In some embodiments, an RNA vaccine or therapeutic may be administered to a subject (e.g., parenterally and mucosally (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal)). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. In some embodiments, an RNA vaccine or therapeutic may be administered intramuscularly. In some embodiments, an RNA vaccine or therapeutic is administered systemically. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous injection, transdermal patches, and the like.

[0344] In some embodiments, the vaccine or therapeutic agent may be administered to a subject at a dose of 1 μg to 100 μg, optionally 8, 10, or 30 μg. The exact amount will depend on the purpose of the treatment and can be ascertained using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0345] In some embodiments, the vaccine or therapeutic may be administered to the subject multiple times. In some embodiments, the vaccine or therapeutic may be administered to the subject at least twice. In some embodiments, the second dose may be administered to the subject about 3 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 8 weeks after the first or first dose. In some embodiments, the second dose may be administered to the subject about 7 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 6 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 5 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 4 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 3 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 2 weeks after the first dose. In some embodiments, the second dose may be administered to the subject about 1 week after the first dose.

[0346] The VZV srRNA vaccine may be administered to a subject, for example, intramuscularly. The vaccine may be administered to a subject at a dose of about 1 μg to about 100 μg, optionally 10 μg or 30 μg. In some embodiments, the vaccine is administered at a dose of at least or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110 μg or more. In some embodiments, the vaccine is administered at a dose ranging from about 1 μg to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 mg. In some embodiments, the vaccine is administered at a dose ranging from about 10 μg to about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 μg. In some embodiments, the vaccine is administered at a dose ranging from about 20 μg to about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 μg. The vaccine may be administered to the subject at least once. The vaccine may be administered to the subject at least twice. The vaccine may be administered to the subject at least three times. The second dose may be administered to the subject about 9 weeks after the first dose. The second dose may be administered to the subject about 8 weeks after the first dose. The second dose may be administered to the subject about 7 weeks after the first dose. The second dose may be administered to the subject about 6 weeks after the first dose. The second dose may be administered to the subject about 5 weeks after the first dose. The second dose may be administered to the subject about 4 weeks after the first dose. The second dose may be administered to the subject about 3 weeks after the first dose. The second dose may be administered to the subject about 2 weeks after the first dose.

[0347] kit

[0348] In some embodiments, the disclosure also provides kits comprising: i) lyophilized RNA; ii) a delivery vehicle, such as a liquid LNP solution; iii) instructions for mixing the lyophilized RNA with the delivery vehicle to prepare an immunogenic composition; and iv) instructions for administering the immunogenic composition to stimulate an immune response to an antigen in a mammalian subject, such as a human subject in need thereof.

[0349] The present disclosure also provides a kit comprising: i) a lyophilized srRNA comprising a VZV gE antigen; ii) a delivery vehicle, such as an LNP; iii) instructions for mixing the first composition with the second composition to prepare an immunogenic composition; and iv) a set of instructions for administering the immunogenic composition to stimulate an immune response against the gE antigen in a mammalian subject, such as a human subject in need thereof.

[0350] In some embodiments, the lyophilized RNA and the liquid LNP solution are stored in separate glass vials. In some embodiments, the liquid LNP solution composition is stored at a temperature between 2-8° C. In some embodiments, the lyophilized RNA composition is stored at room temperature. In some embodiments, the lyophilized RNA composition is stored at a temperature between 2-8° C. In some embodiments, the liquid LNP solution is added to the lyophilized RNA. In some embodiments, the lyophilized RNA and the liquid LNP solution are mixed at room temperature. In some embodiments, the lyophilized RNA and the liquid LNP solution are mixed prior to clinical use.

[0351] definition

[0352] To facilitate the understanding of this invention, a number of terms and phrases are defined below.

[0353] 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. Abbreviations used herein have their conventional meaning within the chemical arts.

[0354] Where systems are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is further contemplated that there are systems of the invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the recited processing steps.

[0355] Whenever an element or component is referred to in this application as being included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or can be selected from a group consisting of two or more of the recited elements or components, or elements or components.

[0356] Furthermore, it should be understood that the elements and / or features of the devices or methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the invention. For example, when a particular component of a system is referenced, that component can be used in various embodiments of the system and / or in the method of the invention, unless otherwise understood from the context. In other words, although embodiments are described and illustrated herein to enable clear and precise description and illustration of the application, it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present teachings and invention. For example, it should be understood that all features described and illustrated herein are applicable to all aspects of the invention described and illustrated herein.

[0357] The articles "a" and "an" are used in this disclosure, unless the context is inappropriate, to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0358] The term "and / or" is used in this disclosure to mean either "and" or "or," unless otherwise indicated.

[0359] The phrase "at least one of" should be understood to include each of the listed objects following this expression individually and various combinations of two or more of the listed objects, unless otherwise understood from context and usage. The phrase "and / or" in the context of more than two listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0360] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, should generally be understood to be open-ended and open-ended, e.g., not excluding additional, unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0361] When the term "about" is used before a quantitative value, the invention also includes the specific, quantitative value itself, unless otherwise specified. As used herein, unless otherwise indicated or inferred from the context, the term "about" refers to a deviation of ±10%, ±5%, ±3%, or ±2% from the nominal value.

[0362] At various places in the specification, variables or parameters are disclosed in groups or ranges. It is specifically intended that the specification include each and every individual subcombination of the members of such groups and ranges. For example, integers in the range of 0 to 40 are specifically intended to disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, and 40 individually, and integers in the range of 1 to 20 are specifically intended to disclose 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 individually.

[0363] The use of any and all examples or exemplary language, such as "such as" or "including" herein is intended merely to better describe the invention and does not limit the scope of the invention unless and until claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0364] As a general matter, formulations specifying percentages are by weight unless otherwise specified. Further, if a variable is not attached to a definition, the previous definition for the variable will control.

[0365] The term "dried RNA" or "dried mRNA" as used herein should be understood as RNA that has been lyophilized, spray-dried, or spray-freeze-dried, as defined herein, to obtain a temperature-stable dry RNA (powder).

[0366] "Cryoprotectants" are well known in the art and include, but are not limited to, sucrose, trehalose, and glycerol. Cryoprotectants that exhibit low toxicity in biological systems are commonly used.

[0367] The term "lyophilization", including the related terms "lyophilization", "lyophilizing", or "freeze-drying", relates to a process that allows for the reduction of the solvent (e.g., water) content of a frozen sample (preferably a solution containing RNA molecules and a cryoprotectant as described herein) in one or more steps, typically by sublimation. In the context of the present disclosure, lyophilization is typically performed by freezing the sample and then drying the sample by sublimation, optionally by reducing the ambient pressure and / or by heating the sample such that the solvent sublimes directly from the solid phase into the gas phase.

[0368] The term "nucleic acid" in its broadest sense includes any compound and / or substance that contains polymers of nucleotides. These polymers can be referred to as polynucleotides.

[0369] The term "messenger RNA" (mRNA) means any polynucleotide that encodes (at least one) polypeptide (natural, non-natural, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in vitro, in vivo, in situ, or ex vivo.

[0370] The term "dose" as used herein with reference to an immunogenic composition means the measured portion of an immunogenic composition taken by (administered to) a subject or received by a subject at any given time.

[0371] The term "immunization" refers to the process of mounting a mammalian subject's response to an antigen, thus improving the subject's ability to resist or overcome infection and / or resist disease.

[0372] As used herein, the term "vaccination" means the introduction of a vaccine into a subject, preferably a mammalian subject, such as a human.

[0373] The term "prophylactically effective dose", including the related terms "effective dose" and "therapeutically effective dose", as used herein means a dose that prevents infection by a virus at a clinically tolerated level.

[0374] The term "alkyl" refers to the radical of a straight or branched chain saturated hydrocarbon group having one to thirty-two carbon atoms ("C1-C 32 In some embodiments, an alkyl group has 1 to 12 carbon atoms ("C-C 12 In some embodiments, an alkyl group has 1 to 10 carbon atoms ("C1-C 10 In some embodiments, an alkyl group has 1 to 9 carbon atoms ("C1-C9 alkyl"). In some embodiments, an alkyl group has 1 to 7 carbon atoms ("C1-C7 alkyl"). In some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1-C5 alkyl"). In some embodiments, an alkyl group has 1 to 4 carbon atoms ("C1-C4 alkyl"). In some embodiments, an alkyl group has 1 to 3 carbon atoms ("C1-C3 alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon atoms ("C1-C2 alkyl"). In some embodiments, an alkyl group has 1 carbon atom ("C1 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). In some embodiments, an alkyl group has 1 to 30 carbon atoms ("C1-C 30 In some embodiments, an alkyl group has 1 to 22 carbon atoms ("C1-C 22 In some embodiments, an alkyl group has 5 to 10 carbon atoms ("C5-C 10 In some embodiments, an alkyl group has 7 to 17 carbon atoms ("C7-C 17 In some embodiments, the alkyl group has 10 to 32 carbon atoms ("C 10 -C 32 "alkyl").

[0375] The term "alkenyl" refers to a radical of a straight or branched chain hydrocarbon group having 2 to 20 carbon atoms, one or more carbon-carbon double bonds and no triple bonds ("C2-C 20 In some embodiments, an alkenyl group has 2 to 10 carbon atoms ("C-C 10 In some embodiments, an alkenyl group has from 2 to 8 carbon atoms ("C2-C8 alkenyl"). In some embodiments, an alkenyl group has from 2 to 6 carbon atoms ("C2-C6 alkenyl"). In some embodiments, an alkenyl group has from 2 to 5 carbon atoms ("C2-C5 alkenyl").

[0376] The term "suitable protective agent" includes the related terms "cryoprotectant" and "protectant" and does not cause or enhance degradation of RNA. EXAMPLES

[0377] Example 1. Preparation of S-2P RNA

[0378] The production of S-2P RNA involved three steps. First, the plasmid DNA encoding the spike glycoprotein of SARS-CoV-2 was amplified, extracted, and then purified by methods well known in the art. Next, the plasmid was linearized by methods well known in the art, and then purified by chromatographic purification and ethanol precipitation. Using the linearized plasmid DNA as a template, RNA was enzymatically synthesized in vitro, followed by purification and storage at -80°C.

[0379] Example 2. Freeze-drying of S-2P RNA

[0380] Lyophilization of S-2P RNA was successfully performed using well-matched protectants and excipients. Briefly, glass vials were filled with 100 μg / mL S-2P RNA (approximately 0.5 mL) in citrate buffer (pH 6) supplemented with approximately 3-12% (w / v) sucrose, such that each vial contained approximately 50 μg of RNA.

[0381] The lyophilization process involves freezing, primary drying, and secondary drying (see detailed setup in Table 1), finally obtaining the RNA in a lyophilized form with very low moisture content (<3%). Like many other lyophilized products, lyophilized RNA can be stored at 2-8°C or at room temperature for extended periods of time without loss of biological activity. [Table 1]

[0382] Example 3. Preparation of lipid nanoparticles

[0383] Lipid nanoparticles (LNPs) were formed or formulated using a microfluidic device by rapidly mixing the ethanol and aqueous phases. The aqueous phase contained 50 mM citrate buffer (pH 5.5). The ethanol phase contained ionizable lipids (as described herein), cholesterol (Jiangsu Southeast Nanomaterials), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Jiangsu Southeast Nanomaterials), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (SINOPEG). A series of ionizable lipids were evaluated. The chemical structures of these lipids are shown in FIG. 1. These four lipid components were mixed in ethanol at a predefined molar ratio, e.g., 48:40.5:10:1.5 (ionizable lipid:cholesterol:DSPC:DMG-PEG2000). The resulting LNP particles were purified in citrate buffer (containing sucrose) to characterize particle size, polydispersity index (PDI), and ionized lipid concentration. The LNP solution was then diluted to the target concentration and stored at 2-8 °C.

[0384] For loaded LNPs, e.g., LNP-encapsulated S-2P RNA liquid drug products, the preparation protocol is essentially the same, except that an aqueous solution of S-2P RNA in citrate buffer at pH 5-6 constitutes the aqueous phase for the LNP formulation. LNP-encapsulated RNA formulations are also referred to as "RNA-LNPs" in this disclosure. With regard to characterization, the particle size and polydispersity index (PDI) of the RNA-LNPs were determined by dynamic light scattering (DLS), RNA encapsulation efficiency and concentration were measured by Ribogreen® assay, and RNA purity was measured by agarose gel electrophoresis.

[0385] Example 4. Preparation and characterization of "ready-to-use" RNA products

[0386] The "ready-to-use" RNA products were prepared by transferring a predetermined amount of already-formed LNPs in solution to a sealed vial containing the lyophilized RNA composition followed by mixing at room temperature. The amount of LNPs added per vial (e.g., 30-100 μg RNA per vial) was determined based on a molar ionized lipid:RNA (N / P) molar ratio of 7. In the present disclosure, "ready-to-use" S-2P RNA formulations (also referred to as "LNPs (S-2P RNA)") containing 30-100 μg / mL S-2P RNA were prepared and subsequently characterized for particle size, polydispersity index, as well as RNA concentration, complexation efficiency, and purity using the same methods as described above in Example 3.

[0387] Example 5. Preparation and characterization of "ready-to-use" S-2P RNA - Proof-of-Concept (PoC) study

[0388] To compare key properties, various batches of LNPs were prepared with ionized lipid (lipid #5 in FIG. 1A) and different molar ratios of ionized lipid:cholesterol:DSPC:DMG-PEG2000 as described in Example 3, all endowed with the same particle size (80±15 nm) and polydispersity index (0.10±0.05). Upon addition of LNPs to the lyophilized S-2P RNA, the lyophilized S-2P RNA was rapidly and completely reconstituted while simultaneously mixing with LNPs, resulting in a homogenous dispersion within seconds. The resulting "ready-to-use" RNA formulation appeared as a colorless, slightly opalescent dispersion, which was identical to its currently commercially available LNP-encapsulated mRNA counterpart. RNA concentrations of at least 100 μg / mL were successfully produced, which is sufficiently high for vaccine applications. Besides appearance, the key characteristics of these two S-2P RNA formulations prepared according to these two different approaches were also compared. As shown in Table 2, the two tested formulations had essentially the same particle size distribution, RNA encapsulation, complexation efficiency, and RNA purity. These data indicate that the novel "ready-to-use" RNA formulations have favorable pharmaceutical characteristics comparable to those of their currently commercially available LNP-encapsulated counterparts. [Table 2]

[0389] Example 6. In vitro transfection

[0390] In the PoC study, 3 x 10 cells were transfected per well to investigate the in vitro transfection efficiency of the S-2P RNA formulation. 5Vero E6 cells were seeded in 6-well plates. 4 μg of the "ready-to-use" RNA formulation formulated with lipid #4 or the LNP-encapsulated S-2P RNA formulation formulated with lipid #9 was transfected into Vero E6 cells, and the spike glycoprotein of SARS-CoV-2 in the cells was detected using Western blot. Briefly, 24 hours after transfection, the cells transfected with the RNA formulations were lysed with NP-40 cell lysis buffer (50 mM Tris, pH 7.4, 150 nM NaCl, 1% NP40, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin). The mixture was centrifuged at 13,000 rpm for 5 min at 4 °C. The supernatants were collected, boiled with SDS at 95°C for 12 min, separated on a 6% SDS-PAGE gel, and transferred to a nitrocellulose filter membrane. After blocking with 5% BSA, the membranes were first blotted with a primary antibody (1:1000) (SARS-CoV-2 (2019-nCoV) spike rabbit PAb, 40592-T62, Sino Biological), and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000) (IgG (H+L) (HRP-labeled goat anti-rabbit IgG (H+L))), Beyotime). Finally, they were visualized with a chemiluminescent reagent (chemiluminescent HRP substrate, WBKLS0500, Millipore). 24 hours after transfection, the spike glycoprotein of SARS-CoV-2 in the cells was detected using Western blot. Based on the S-2P protein expression levels shown in FIG. 2, the "ready-to-use" RNA formulations described herein are at least as effective as the widely available LNP-encapsulated RNA formulations.

[0391] In a formulation screening study (Example 3), the in vitro transfection efficiency of test articles of "ready-to-use" S-2P RNA was investigated in both BHK-21 and Vero E6 cell lines. Briefly, 3 x 10 per well was transfected with 100% PBS. 5Vero E6 or BHK-21 cells were seeded in 6-well plates. Cells were transfected with 4 μg of each investigated "ready-to-use" S-2P RNA test article. 48 h after transfection, RBD expression in cell culture supernatants was quantified with a commercial SARS-CoV-2 (2019-nCoV) spike RBD ELISA kit (KIT40592, Sino Biological) according to the manufacturer's instructions. Supernatants were diluted 200-fold. The final concentration of RBD was calculated based on a linear standard curve of absorbance at 450 nm. Briefly, detection wells were pre-coated with a monoclonal antibody specific for the spike RBD protein. Samples or standards were incubated for 2 h at room temperature (RT), after which samples not bound to the immobilized antibody were removed by a washing step. Then, detection antibody was added for 1 h incubation at room temperature. After washing, the substrate solution was added to each well while blocking light. The stop solution was added to each well after 20 minutes, and the absorbance at 450 nm was measured. The results are shown in Table 4.

[0392] Example 7. In vivo evaluation of "ready-to-use" S-2P RNA

[0393] Animal studies were performed at the Yangtze Delta Region Research Institute of Tsinghua University (Zhejiang). BALB / c mice (6-8 weeks old) were grouped with n=4. On day 0 (prime injection) and day 21 (boost), three groups of mice were immunized intramuscularly with the "ready-to-use" S-2P RNA formulation (8 μg) or the LNP-encapsulated S-2P RNA formulation (5 μg) and buffer vehicle, respectively. For the "ready-to-use formulation", the formulation used was 48:40.5:10:1.5 (lipid#4:cholesterol:DSPC:DMG-PEG2000). For the lipid-encapsulated formulation, the formulation used was 40:48.5:10:1.5 (lipid#9:cholesterol:DSPC:DMG-PEG2000). Serum was collected before the first vaccination, as well as on days 13, 20, 28, and 35. All collected samples were cryopreserved according to standard protocols (Figure 3).

[0394] Example 8. Quantification of antibody binding titers to SARS-CoV-2 spike protein by ELISA assay

[0395] Antibody binding titers against SARS-CoV-2 spike protein (RBD, Histag) (GenScript Z03483) were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 0.5 μg / mL of SARS-CoV-2 spike protein (RBD, Histag) diluted in carbonate-bicarbonate buffer was precoated onto 96-well clear polystyrene microplates (Corning) overnight at 4 °C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plates were blocked with 300 μL of blocking buffer (15% normal goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37 °C. Serum samples were serially diluted 2-fold in blocking buffer, transferred to the plates, and incubated at 37 °C for 1 h. After washing, the plates were incubated with HRP-conjugated rabbit anti-mouse IgG H+L (Abcam, ab6728) for 1 hour at 37° C. The plates were washed and incubated with TMB single component substrate solution (Solarbio, PR1200) for 7 minutes at 37° C., and the reaction was stopped with ELISA stop solution (Solarbio, C1058). Absorbance was read at 450 nm on a microplate reader (VARIOSKAN LUX, ThermoFisher) and ELISA titers were determined using a nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software (FIG. 4).

[0396] Example 9. Screening for LNP compositions suitable for "ready-to-use" RNA products

[0397] Two formulation variables, i) the structure of the ionizable lipids; and ii) the lipid component ratio, were investigated in a series of experiments to identify a range of LNP compositions. Other relevant formulation parameters, such as pH and N / P, utilized in the manufacturing process were also investigated.

[0398] All formulations investigated after the screening study were prepared according to the standard protocol described in Example 3 for comparison purposes without further optimization of the formulation or manufacturing process parameters, respectively.

[0399] Example 10. Structure of ionized lipids

[0400] To investigate the effect of ionizable lipid structure on the pharmaceutical profile of the product, a series of blank LNPs (i.e., no RNA) were prepared using 12 different ionizable lipids (see Figures 1A-1B for chemical structures) each along with three other lipid components (DMG-PEG, DSPC, and cholesterol) following the protocol described in Example 3. Although composed of different ionizable lipids, these 12 blank-LNP formulations used the same lipid molar ratio as utilized in Example 3, i.e., 48:40.5:10:1.5 (ionizable lipid:cholesterol:DSPC:DMG-PEG2000).

[0401] Each resulting blank-LNP formulation was added and mixed with lyophilized S2P RNA, together resulting in a series of "ready-to-use" S2P RNA test articles containing different ionizable lipids. The pharmaceutical characteristics (Table 3) and in vitro transfection efficiency (Table 4) of these "ready-to-use" RNA test articles were directly compared. In parallel, the stability profiles of these 12 blank-LNP formulations (i.e., without RNA complexation) were investigated at 2-8°C (see Table 5). [Table 3] Test articles were prepared with a fixed lipid molar ratio (48 mol% ionizable lipid / 40.5 mol% cholesterol / 10 mol% DSPC / 1.5 mol% DMG-PEG). In all cases, 0.6 mL of blank-LNP was added to lyophilized S2P RNA (55 μg / vial) at a molar ratio of ionizable lipid:RNA (N / P) of 7.

[0402] [Table 4]

[0403] [Table 5] Test articles were prepared with a fixed lipid molar ratio (48 mol% ionizable lipid / 40.5 mol% cholesterol / 10 mol% DSPC / 1.5 mol% DMG-PEG).

[0404] As shown in Table 3, the particle size and PDI of the resulting "ready to use" RNA test articles were affected by the choice of ionized lipid but still fell within the acceptable range for the intended application, e.g., after intramuscular administration. However, the RNA complexation efficiency readout varied across the 12 test articles, with formulations containing lipids #2 and #3 being the highest compared to the other formulations tested. This data indicates that "ready to use" RNA products with sufficiently high RNA complexation efficiency can be obtained with ionized lipids structurally similar to lipids #1-#6 (as shown by RNA complexation efficiency of over 40% in this experiment without optimization of formulation and process parameters).

[0405] The in vitro transfection efficiency of these 12 "ready-to-use" S2P RNA test articles was compared in BHK-21 as well as Vero E6 cell lines at equal doses (4 μg). As evidenced by the expression levels (Table 4), the test articles containing ionized lipids #4 / #5 / #6 yielded the highest transfection efficiency, followed by the test article containing lipid #2.

[0406] Besides the pharmaceutical characteristics and transfection efficiency, the ionized lipid structure may also affect the stability profile of blank-LNP. As shown in Table 5, these 12 formulations showed a wide range of stability profiles at 2-8°C by incorporating different ionized lipids. Among all the tested items, LNP test items containing lipids #2, #3, #4, #5, or #8 showed no change in size and quantifiable stability upon storage at 2-8°C for one month.

[0407] Taking into account all the evaluations tested herein, lipids #4 and #5 are the superior ionizable lipids among all the structures tested, followed by lipid #2. Ionizable lipids with these structures appear to provide blank-LNPs with quantifiable stability at 2-8°C, and upon mixing with lyophilized RNA, generate "ready-to-use" RNA formulations with favorable pharmaceutical characteristics and in vitro transfection efficiency. Ionizable lipids #4, #5, and #2 have in common 4-N,N dimethylamino-butanoate, 2-methyl-1,4-phenylenedioxyl linkers, alkyl tails independent of octanoate, and C18-C26 alkyl tail head groups, which are examples of formula (II).

[0408] Example 11. Molar fraction of lipid components constituting LNPs

[0409] The LNPs described herein generally comprise four lipid-based components: 1) an ionizable lipid, 2) a phospholipid (e.g., DSPC), 3) cholesterol, and 4) a PEGylated lipid (e.g., DMG-PEG2000). Each of these components, as well as their relative ratios, can play a role in the composition of the LNP. To develop a "ready-to-use" RNA formulation, LNPs composed of different ratios of lipids were screened to identify lipid compositions suitable for a "ready-to-use" RNA product.

[0410] To investigate the boundaries of the ratio of each lipid component, we first synthesized 14 blank-LNP formulations containing the same lipid components at different feeding ratios. As shown in Table 6, in formulations 1-6 and 8, the amount of DMG-PEG was varied between 0.25 and 4 mol% while other lipid components were fixed (except for minor ratio changes of cholesterol, if necessary). In formulations 7-9, the amount of DSPC was adjusted between 5 and 20 mol% while the amount of cholesterol was concomitantly changed. In formulations 10-12, 8, 13, and 14, the amount of ionizable lipid was increased from 20 to 70 mol% while the amount of cholesterol was decreased from 68.5% to 18.5 mol%, as appropriate. This experimental design investigated the mole fractions of lipid components that, independently or in combination, affect the formation and pharmaceutical characteristics of LNPs.

[0411] [Table 6]

[0412] The 14 resulting blank-LNP formulations were characterized in terms of particle size and PDI both at the end of manufacturing (T=0) and upon 8 weeks of storage at 2-8° C. As shown in Table 6, this pilot screening work provides typical ratio ranges for each lipid component that allow obtaining LNPs with generally favorable particle size (≦150 nm), PDI (≦0.25), and stability profile at 2-8° C. (defined as a size increase of ≦20 nm over 8 weeks), i.e., at least 1-4 mol % DMG-PEG2000, at least 5-20 mol % DSPC, at least 30-70 mol % ionizable lipid, and at least 18.5-58.5 mol % cholesterol. A series of LNP formulations using two structurally diverse ionizable lipids, namely lipids #4 and #6, were prepared. In this experiment, the fraction of each ionizable lipid was set at 20-70 mol% of the total lipid, while the fractions of the other lipid components were allocated outside the allowed ranges described in the paragraph above. The pharmaceutical characteristics of the blank-LNP and the resulting "ready-to-use" RNA formulations, as well as the stability profile (2-8°C) of the blank-LNP test article, were evaluated.

[0413] With ionizable lipid #4, a molar fraction of 30-60 mol% results in not only an acceptable size distribution but also a relatively large RNA complexation efficiency (above 40%), as shown in Table 7. For ionizable lipid #6, the maximum RNA complexation efficiency was achieved when the molar fraction was kept between 30-50 mol%. [Table 7]

[0414] Besides the pharmaceutical characterization, a direct comparison of these "ready-to-use" S2P RNA test articles was also performed with respect to transfection efficiency in BHK-21 and Vero E6 cell lines at equal doses (4 μg). Besides determining RBD expression levels (Table 8, measured by ELISA), it was found that the fraction of ionized lipids at 30-60 mol% worked optimally for transfection of the "ready-to-use" RNA formulations in cells in this study. [Table 8]

[0415] With respect to the stability profile at 2-8° C., the blank-LNP test items containing ionized lipid #4 were overall more stable than those containing lipid #6. As shown in Table 9, LNPs containing lipid #4 showed good stability at 2-8° C. as long as the fraction of ionized lipid was kept below 60 mol %. [Table 9]

[0416] Based on all experimental evaluations, the ionized lipid fraction of 30-60 mol% is the best for conferring not only favorable pharmaceutical and biological performance of the "ready-to-use" RNA product, but also for providing a long shelf-life of the blank-LNP lipid product at 2-8° C. Regarding the other lipid components, it was shown that 5-20 mol% DSPC / 1-4 mol% DMG-PEG / 18.5-58.5 mol% cholesterol seems to be well suited for generating the "ready-to-use" RNA product.

[0417] Example 12. pH of LNP dispersion formulation added to lyophilized RNA

[0418] In LNP formulations, the ionization state of ionizable lipids can be determined by the pH of the aqueous dispersion. In these embodiments, the pH of the blank-LNP dispersion (and therefore of the "ready-to-use" RNA formulation) can affect the complexation efficiency of anionic RNA. In an acidic environment, RNA complexation is favored at a pH below the pKa value (i.e., the negative base-10 logarithm of the acid dissociation constant) of the LNP liquid formulation.

[0419] The pKa of blank-LNPs composed of ionized lipids #1-12 is generally 6.0-6.5. To provide pH range considerations for these embodiments, three blank-LNP formulations containing the same lipid composition but dispersed in buffers at different pHs (pH 5.0, pH 5.5, and pH 6.0) were synthesized using lipid #4 and used to prepare three "ready to use" S2P RNA test items according to the following protocol. The LNP solution for the S2P RNA formulation was gently shaken for approximately 5-10 seconds prior to use. Then, 1.0 mL of the solution was aspirated using a needle syringe and slowly added to the lyophilized composition along the wall of the vial. After complete injection, the solution was mixed by shaking upside down for approximately 30 seconds. After mixing, the solution appeared as a milky to white suspension. After mixing and placing at room temperature for approximately 1 hour, approximately 0.8 mL of the S2P RNA formulation solution was aspirated using a syringe. As shown in Table 10, LNP dispersions at pH 5.0-6.0 were found to be suitable for RNA complexation in these embodiments and under the conditions tested. [Table 10] Test articles were prepared with a fixed lipid composition (48 mol % ionized lipid #4 / 40.5 mol % cholesterol / 10 mol % DSPC / 1.5 mol % DMG-PEG).

[0420] Example 13. Ratio of RNA to Ionizable Lipid, expressed as Ionizable Lipid:RNA (N / P) Molar Ratio

[0421] For LNP-encapsulated RNA formulations, an ionizable lipid:RNA (N / P) molar ratio of about 6 is often utilized (Int J Pharm. 2021 May 15;601:120586). To explore the range of N / P ratios suitable for these embodiments, a desired amount of blank-LNP formulation (pH 5.5) was added to lyophilized S2P RNA based on a given ionizable lipid:RNA ratio of 5, 7, or 9 to generate a "ready to use" RNA formulation.

[0422] The effect of N / P molar ratio on particle size, PDI, and RNA complexation efficiency of the resulting "ready to use" RNA test articles was characterized. As shown in Table 11, high N / P promoted the formation of "ready to use" RNA products with high RNA complexation efficiency and small hydrodynamic size. N / P molar ratios greater than 5 and less than 9 appeared to be suitable for these embodiments under the test conditions.

[0423] Table 12 summarizes the composition and parameters of the "ready-to-use" RNA products. [Table 11] Test articles were prepared with a fixed lipid composition (48 mol % ionized lipid #4 / 40.5 mol % cholesterol / 10 mol % DSPC / 1.5 mol % DMG-PEG). [Table 12]

[0424] Example 14. VZV glycoprotein E (gE) antigen

[0425] VZV glycoprotein E (gE) was selected as a candidate vaccine antigen. VZV gE is a type I membrane protein of 623 amino acids that contains a signal peptide, the major part of the protein, a hydrophobic anchor domain, and a C-terminal tail. The C-terminal domain of VZV gE contains a Y569A mutation that can regulate cellular trafficking to the trans-Golgi network (TGN) and expression of the VZV gE protein [1]. The srRNA vaccine construct contains RNA encoding the 573 amino acid carboxyl-terminal truncated VZV gE protein encoded by SEQ ID NO:3 (Oka strain).

[0426] Example 15. RNA in vitro transcription and capping

[0427] The self-replicating RNA (srRNA) backbone was based on a recombinant alphavirus genome containing genes encoding nonstructural proteins that enable RNA replication. The structural protein sequences were replaced with the gene sequence of VZV glycoprotein E. The srRNA vaccine construct contains a 5' capped untranslated region (UTR), four nonstructural genes (nsp1-4), a 26S subgenomic promoter, a VZV glycoprotein E gene, and a 3' terminal polyadenylation tail encoded by SEQ ID NO: 4 (Figure 9). To generate a linear template for RNA transcription, the plasmid DNA was cleaved by restriction digestion with BspQI enzyme (New England Biolabs, R0712L) and purified using the PureLink® PCR Purification Kit (Invitrogen, K310002). RNA was transcribed using 5000U / mL T7 polymerase (New England Biolabs, M0251L), 1000U / mL RNase inhibitor (New England Biolabs, M0314L), 2 / mL pyrophosphatase (New England Biolabs, M2403L), 2mM spermidine (Sigma, 5292), 10mM DL-dithiothreitol (Sigma, 43816), 6mM rNTPs (New England Biolabs, N0466S), 24mM MgCl2 (Invitrogen, AM9530G), and 40mM Tris-HCl (Sigma, T2694). The mixture was incubated and shaken at 37°C for 3 hours. RNA transcripts were capped with variola capping enzyme (New England Biolabs, M2080S) and mRNA cap 2'-O-methyltransferase (New England Biolabs, M0366) using GTP (Invitrogen, R0461) and S-adenosyl-methionine (New England Biolabs, B9003S) as substrates to generate the cap-1 structure. RNA was purified using LiCl precipitation.

[0428] Example 16. Preparation of formulation

[0429] Microfluidic Device (INano TM Lipid nanoparticles were formulated by rapidly mixing the ethanol and aqueous phases using a 300-mL system (Micro&Nano). The aqueous phase was 50 mM citrate buffer (pH 6) containing purified srRNA. The ethanol phase contained the ionizable lipid, heptadecan-9-yl 8-(3-(((4-(dimethylamino)butanoyl)oxy)methyl)-4-((8-(nonyloxy)-8-oxooctyl)oxy)phenoxy)octanoate 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti, 850365P), cholesterol (Sigma-Aldrich, C8667), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (NOF, GM020). RNA-LNPs were assembled at a molar ratio of 10:48:2:40 (DSPC:cholesterol:PEG 2000:LKY-XH15) with a NP lipid:RNA ratio of 8. The formulations were characterized for particle size, RNA concentration, encapsulation efficiency, and protection ability from RNase digestion.

[0430] Example 17. Mode of Vaccine Administration

[0431] All animal studies were performed at Fangcheng Gang Lab of Shanghai HkeyBio Technology Co.,Ltd. (Fangcheng Gang Spring Biological Technology Development Corporation Ltd,China), and all experiments involving laboratory animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Fangcheng Gang Spring Biological Technology Development Corporation Ltd. To mimic natural VZV infection, two groups of three adult male cynomolgus macaques were immunized subcutaneously (SC) once with live attenuated VZV (LAV) vaccine 28 days (day -28) before the start of the study. On days 0 and 62, the monkeys were immunized SC once with 10-30 μg VZV gE RNA / LNP, respectively (Figure 5). Serum and peripheral blood mononuclear cells (PBMCs) were collected 14 days after vaccination, and the final collection was performed 5 months after the second vaccination. All samples were stored frozen according to standard protocols.

[0432] Example 18. ELISA and binding activity assays

[0433] Antibody binding titers against VZV gE from monkeys were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 0.5 μg / mL of recombinant VZV gE protein (Abcam, ab43050) diluted in carbonate-bicarbonate buffer was precoated onto 96-well clear polystyrene microplates (Corning) overnight at 4°C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plates were blocked with 300 μL of blocking buffer (15% goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37°C. Serum samples were serially diluted two-fold in blocking buffer, transferred to the plates, and incubated for 1 h at 37°C. After washing, the plates were incubated with HRP-conjugated goat anti-monkey IgG H+L (Abcam, ab112767) for 1 hour at 37°C. The plates were washed and incubated with TMB single component substrate solution (Solarbio, PR1200) for 7 minutes at 37°C, and the reaction was stopped with ELISA stop solution (Solarbio, C1058). Absorbance was read at 450 nm on a microplate reader (SpectraMax Abs Plus, Molecular Devices). ELISA titers were determined using a nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software. Data not reaching the EC50 were set to a baseline value of 10 because extrapolation beyond the data curve is inaccurate (Figure 6).

[0434] To evaluate the binding activity of the antibodies, ELISA was performed as described above with minor modifications, if necessary. After serum incubation, the plates were washed three times with PBS-T and incubated with 8M urea (TCI, U0073) diluted in PBS for 5 minutes at room temperature. The plates were washed three times with PBS-T and the remainder of the ELISA was performed as described above. The binding activity index was calculated as the EC50 of the wells treated with 8M urea divided by the EC50 of the control wells without urea treatment (Figure 7).

[0435] Example 19. Stimulation of PBMC cells and intracellular cytokine staining

[0436] Cryopreserved cynomolgus monkey PBMCs were quickly thawed in a 37°C water bath and washed with medium (RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1x penicillin-streptomycin). Cells were distributed into 96-well round-bottom plates and restimulated in vitro with 2 μg / mL of VZV gE protein (15-mer, overlapping 11aa) and 1.25 μg / mL of a pool of peptides spanning CD28 monoclonal antibody (Invitrogen, 16-0289-81) and CD49d monoclonal antibody (Invitrogen, 16-0499-81). Plates were incubated at 37°C, 5% CO2 for 2 hours, followed by overnight treatment with protein transport inhibitor cocktail (Invitrogen, 00-4980-93). Cells were washed with Dulbecco's phosphate-buffered saline (D-PBS) and LIVE / DEAD TM Fixed aqueous dead cell staining (LIVE / DEAD TM The cells were stained with fixable aqua dead cell stain (Invitrogen, L34965) for 30 min. The cells were washed with FACS wash buffer (D-PBS containing 2% FBS) and incubated with fluorescently labeled antibodies for 30 min to stain cell surface proteins. The antibodies were mouse anti-human CD3 APC-Cy TMAntibodies used included CD4 Brilliant Violet 421 (clone L200), and CD8 FITC (clone RPA-T8) (all from BD Biosciences). Cells were washed with FACS wash buffer and incubated with Fixation / Permeabilization Standard Solution (Invitrogen, 00-51232-43) for 30 minutes according to the manufacturer's instructions. Cells were washed twice with Permeabilization Buffer (Invitrogen, 00-8333-56) and incubated with fluorescently labeled antibodies for 30 minutes to detect cytokine expression within the cells. Antibodies included mouse anti-human IFN-γ PE (clone 4S.B3 from BD Biosciences) and TNF-α PerCP (clone MAb11), as well as rat anti-human IL-2 (clone MQ1-17H12) (all from Biolegend). Cells were washed with permeabilization buffer and resuspended in MACSQuant running buffer (Miltenyi Biotec). Fluorescent signals were acquired using MACSQuant16 (Miltenyi Biotec). Data were analyzed with FlowJo software (version X) (Figure 8).

[0437] Example 20. Efficacy of srRNA vaccine constructs in cynomolgus monkeys

[0438] As outlined above, cynomolgus macaques (3 monkeys per group) were inoculated with LAV followed by 10 μg or 30 μg of srRNA vaccine-encoded VZV gE by intramuscular delivery on both days 0 and 62 (FIG. 5).

[0439] VZV gE-specific antibody binding titers were quantified by VZV gE ELISA. As shown in Figure 6, VZV gE-specific antibody titers were relatively low after LAV inoculation, but the srRNA vaccine induced high and sustained VZV gE-specific antibody responses after two immunizations. Furthermore, a dose-escalation effect was observed across srRNA vaccine doses. The avidity of antibodies in the serum of immunized monkeys was also determined and is shown in Figure 7. The avidity index indicates that both 10 μg and 30 μg srRNA vaccine immunizations resulted in rapid antibody affinity maturation (Figure 7).

[0440] To measure the frequency and magnitude of antigen-specific T cell subsets, PBMCs from immunized cynomolgus macaques were stimulated with peptides spanning the VZV gE protein and response capacity was examined by production of IFN-γ and TNF-α, as well as IL-2, in CD4+ and CD8+ T cells. Individual cytokine expression data are shown in Figure 8. Four weeks after immunization 3 (day 90), the srRNA vaccine significantly increased and induced high frequencies of VZV gE-specific CD4+ T cells and much more potent CD8+ T cells.

[0441] Compared to Shingrix and conventional (non-replicating) mRNA, the srRNA vaccine induces substantial levels of antibody responses, stronger CD4+ T cell responses, and much stronger CD8+ T cell responses, whereas Shingrix and conventional mRNA do not induce detectable CD8+ T cell responses. This striking difference in CD8+ T cells was unexpected and provides an advantage over the current standard of care.

[0442] Example 21. Relative potency of lyophilized RNA

[0443] Lyophilized self-replicating RNA encoding a model protein (green fluorescent protein, GFP) was stored at approximately 4° C. or at room temperature (approximately 20-24° C.). Data are shown in FIG. 10A and FIG. 10B. Samples were taken at selected time points and tested using an in vitro potency assay that compares the total intensity of the fluorescent signal emitted by baby hamster kidney cells, when transfected with a mixture of equal amounts of RNA and Lipofectamine 2000 and cultured for 48 hours, to a reference standard. The relative potency at time 0 was set to 100%.

[0444] Example 22. VZV srRNA vaccine - animal studies

[0445] Prior to administration, the srRNA component in the vial and the LNP dispersion in a separate vial were allowed to equilibrate to room temperature for approximately 15 minutes. The LNP dispersion was gently shaken for 5-10 seconds. Using a needle-syringe combination, 0.6 mL of the LNP dispersion was withdrawn from the vial and added to the lyophilized srRNA in the first vial. The vial was then inverted upside down for approximately 30 seconds to thoroughly mix, yielding the reconstituted vaccine, which appeared as a white to off-white suspension.

[0446] Animal studies were performed using 80 female SPF C57BL / 6 mice that were randomly assigned into seven groups (Table 13) and immunized with the test articles according to a predetermined immunization schedule, as shown in the schematic diagram in FIG. 11. [Table 13]

[0447] To mimic natural VZV infection and subsequent latency, mice in groups (G) 3-6 were pre-immunized subcutaneously (sc) with live attenuated influenza virus (LAV) vaccine (3.3 lg PFU) in the nape of the neck 35 days (day -35) prior to the start of the study. Mice in G1, G3, and G6 were immunized twice by intramuscular (im) administration of VZV srRNA vaccine at 15 μg per dose (G1, G3) or 3 μg per dose (G6) in the quadriceps of the hind legs on days 0 and 28, respectively. In groups G2 and G4, mice were immunized once intramuscularly with VZV srRNA vaccine at 15 μg per dose on day 28. In the saline control group (G5), mice were administered sterile saline intramuscularly on days 0 and 28. Intramuscular (im) injections were performed in the quadriceps of the hind limbs at multiple sites, not exceeding 0.1 mL per site. In the natural control group (G7), mice were maintained without any treatment. Detailed information on the grouping and treatment of mice is shown in Figure 13.

[0448] At the time points indicated in the vaccination schedule (Figure 7), blood samples (50-100 μL per mouse) were collected and serum preparation was performed as described in the protocol. Spleens were harvested from 50% of the mice in each group on day 42 for splenocyte isolation, as per the protocol. The other 50% of the mice in the study were kept for tracking of immune responses.

[0449] Serum preparation

[0450] Blood samples were collected in Eppendorf tubes and kept on ice. After centrifugation at 1,500 g for 10 min at 4°C, the supernatants were quickly transferred to new tubes and stored below -70°C.

[0451] Isolation of splenocytes

[0452] To isolate splenocytes, fresh spleens were isolated from immunized female SPF C57BL / 6 mice or control mice and gently homogenized in PBS using a piston syringe. The cell suspension was then passed through a 70 μm cell strainer (BD Falcon). Red blood cells were lysed using red blood cell lysis buffer according to the manufacturer's instructions (Invitrogen). After washing twice with PBS, the cells were suspended in 0.5 mL of medium (RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1× penicillin-streptomycin).

[0453] Enzyme-linked immunosorbent assay (ELISA)

[0454] Antibody titers against VZV gE were quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, 0.5 μg / mL of recombinant VZV gE protein diluted in ELISA-containing buffer was precoated on 96-well clear polystyrene microplates overnight at 4° C. After washing three times with 0.5% PBS-T, the coated plates were blocked with 300 μL of ELISA blocking buffer per well for 1 h at 37° C. Serum samples were serially diluted two-fold in blocking buffer, transferred to the coated plates, and incubated for 1 h at 37° C. After washing, the plates were incubated with HRP-conjugated rabbit anti-mouse IgG (H+L) antibody for 1 h at 37° C. The plates were washed three times with 0.5% PBS-T and incubated with TMB single component substrate solution for 7 min at 37° C. The reaction was then stopped with ELISA stop solution. Absorbance was read at 450 nm in a microplate reader. To determine antibody levels, the half maximal effective concentration (EC50) of each sample was calculated using a nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software. The EC50 value was used to represent the antibody level for each sample.

[0455] cell stimulation

[0456] Fresh splenocytes (2x106 in 200μL medium) were seeded into 96-well round-bottom microplates and restimulated with 1.25μg / mL of VZV gE protein (pool of 138 15-mer peptides with 11 amino acid overlap as listed in Appendix 7.6) and 1.25μg / mL of pools of peptides spanning CD28 and CD49d monoclonal antibodies. Plates were incubated at 37°C in a humidified incubator with 5% CO2 for 2 hours, followed by overnight treatment with protein transport inhibitor cocktail.

[0457] Cell surface and intracellular staining

[0458] The cells were washed with Dulbecco's phosphate buffered saline (DPBS) and LIVE / DEAD TM Fixed aqueous dead cell staining (LIVE / DEAD TM The cells were stained with fixable aqua dead cell stain (FACS) for 30 minutes. The cells were washed with 200 μL of fluorescence-activated cell sorting (FACS) wash buffer and incubated with fluorescently labeled primary antibodies for 30 minutes to stain cell surface proteins. Antibodies included anti-mouse CD3 APC-Vio 770 (clone REA641), anti-mouse CD4 VioBlue (clone REA604), and anti-mouse CD8 PerCP (clone REA601) (1 μL per well). The cells were then washed with FACS wash buffer and incubated with fixative / permeabilization standard solution for 30 minutes according to the manufacturer's instructions. The cells were then washed twice with permeabilization buffer and incubated with fluorescently labeled primary antibodies for 30 minutes to detect cytokine expression in the cells. Antibodies included anti-mouse IFN-γ FITC (clone REA638), anti-mouse TNF-α PE (clone REA636), and anti-mouse IL-2 APC (clone REA665) (1 μL per well). Finally, cells were washed with 200 μL of permeabilization buffer and resuspended in 200 uL of PBS for flow cytometry analysis.

[0459] Sample analysis

[0460] Stained cells were analyzed using a CytoFlex flow cytometer (Beckman). Data was analyzed using CytExpert software.

[0461] VZV gE-specific humoral immune responses

[0462] Humoral responses were determined by VZV gE-specific IgG antibody binding titers, which were quantified by ELISA on serum samples taken at the indicated time points after immunization. Antibody levels were expressed as EC50 values ​​determined as described.

[0463] In this study, we used the LAV-primed mouse model to evaluate the immunogenicity of the VZV srRNA vaccine. The data are shown in Figure 12. LAV-primed mice injected with sterile saline (G5) showed a slight increase in VZV gE-specific IgG antibody levels, 2-fold (p<0.0001) on day 0 and 3-fold (p<0.0001) on day 14, but similar on days 28 and 42 compared to the natural control group (G7). After LAV-primed immunization, antibody levels were 105-fold higher (p<0.05) in the single-dose VZV srRNA vaccine group (G4) than in the non-vaccine immunization group (G5) on day 42. Meanwhile, in the two-dose immunization groups, antibody levels at day 14 after the first immunization were also higher at 15 μg per dose (G3) and 3 μg per dose (G6) compared to G5 (70-fold; p<0.0001 and 46-fold; p<0.0001, respectively). The humoral response was further improved after the second immunization, as shown by significantly higher antibody levels at day 42 in G3 and G6 than in G5 (332-fold; p<0.0001 and 271-fold; p<0.0001, respectively). It was noteworthy that antibody levels induced by the high dose (15 μg) were similar to those at the low dose (3 μg) at days 14 (2.5-fold; p=0.241), 28 (2.3-fold; p=0.829), and 42 (1.6-fold; p=0.530). Taken together, in the presence of LAV preimmunization, either a single or two-dose vaccination induced strong VZV gE-specific humoral immune responses. Furthermore, VZV srRNA vaccination at a low dose (3 μg per dose) induced similar antibody levels as at a high dose (15 μg per dose) in mice.

[0464] To further evaluate the immunogenicity of VZV srRNA vaccine in the absence of LAV priming, the effects of either a single or two-dose vaccine immunization at 15 μg per dose on humoral responses were compared. Compared with the natural control group (G7), one VZV srRNA vaccine immunization induced a moderate upregulation of VZV gE-specific antibody levels, 15-fold (p<0.0001) on day 14 in G1 and 17-fold (p<0.0001) on day 42 in G2, respectively. After the second immunization, humoral responses were significantly improved, with antibody levels on day 42 in G1 being 197-fold higher than those in G7 (p<0.0001). Furthermore, the effect of LAV priming on VZV gE-specific IgG antibody levels induced by VZV srRNA vaccine immunization was investigated. In the two-dose VZV srRNA vaccine immunization groups (G1 and G3), the antibody levels in G3 were higher than those in G1 on days 14 (14-fold; p<0.0001), 28 (3.4-fold; p<0.0001), and 42 (2.9-fold; p<0.01), respectively. In the single-dose immunization groups (G2 and G4), the VZV gE antigen-specific IgG antibody response in G4 was higher than that in G2 on day 42 (9.6-fold; p<0.05). These results suggested that LAV priming affected the levels of gE-specific IgG antibodies induced by the VZV srRNA vaccine immediately after one immunization, but the effect of LAV prepriming was less evident after the second VZV srRNA immunization at the high dose. Thus, administration of a VZV srRNA vaccine, in the absence of LAV priming, can also induce significant VZV gE-specific humoral immune responses.

[0465] VZV gE-specific cellular immune responses

[0466] VZV antigen-specific T cell responses are important for recovery from primary VZV infection and for preventing reactivation of latent VZV. Therefore, the frequencies of antigen-specific T cell subsets were measured in splenocytes of immunized mice after in vitro restimulation with a peptide pool spanning the VZV gE protein.

[0467] First, we evaluated the effect of LAV priming on VZV gE-specific cellular immune responses. Compared with the natural control (G7), the frequency of total cytokine (IFN-γ and / or TNF-α)-expressing CD4+T or CD8+T cells induced in the LAV-primed control group (G5) was not significantly increased on day 42 (p=0.034, p=0.181, respectively). Thus, administration of LAV alone was not able to activate VZV gE-specific T cell immune responses in mice (Figures 13A and 13B).

[0468] Immunization with two doses of VZV srRNA vaccine after LAV priming significantly increased the frequency of VZV gE-specific CD4+ T cells expressing IFN-γ and / or TNF-α compared to the LAV-primed control group (G5) without VZV srRNA injection (6.1-fold increase in G3, p<0.0001 and 7.5-fold increase in G6, p<0.0001) (Figure 14). Furthermore, CD4+ T cells in G6 were similar to those in G3 (p=0.122), indicating that a low dose of VZV srRNA vaccine was sufficient to stimulate a strong cellular immune response. Moreover, similar to two doses of VZV srRNA vaccine, the frequency of total cytokine (IFN-γ and / or TNF-α)-expressing GD4+T in G4 was 5.2-fold higher than that in the control group (G5), suggesting that immunization with a single dose of VZV srRNA vaccine could also significantly activate CD4+T cells. These results demonstrated that a significant VZV gE-specific CD4+T cell immune response was activated by either a single or two doses of VZV srRNA vaccine after pre-immunization with LAV.

[0469] Finally, we investigated the effect of LAV priming on the cellular immune response activated by VZV srRNA vaccine immunization. In the two-dose VZV srRNA vaccine immunization groups with and without LAV priming, the frequency of total cytokine (IFN-γ and / or TNF-α)-expressing CD4+ T cells in G1 was similar to that in G3 (p=0.576). In the single-dose immunization groups (G2 and G4), the frequency of total cytokine (IFN-γ and / or TNF-α)-expressing CD4+ T cells in G2 without LAV priming was slightly higher than that in G4 with LAV priming (p=0.044). Thus, administration of VZV srRNA vaccine in the absence of LAV priming could also induce VZV gE-specific CD4+ T cell immune responses (Figure 14).

[0470] Example 23. srRNA structures with extended 3'UTR and / or polyA tails

[0471] In this example, we examine modifications of the 5' cap structure, modulation of the length of the poly(A) tail, including modified nucleotides, codons, or sequence optimization, as well as changes in the 5' and 3' UTRs to improve srRNA structure.

[0472] Native eukaryotic mRNAs have a 7-methylguanosine (m7G) cap attached to the mRNA. The 5'UTR capping of IVT mRNAs contains at least three forms: cap0 [m7G(5')pppN1pN2p], cap1 [m7G(5')pppN1mpNp], and cap2 [m7G(5')pppN1mpN2mp]. Alphavirus 7-methylguanosine (m7G) cap structure (cap0) capping is in some fashion promoted by nsP1 and lacks this additional 2-methylation modification. The length of the 5' and 3'UTRs varies among alphaviruses, with SIN 5'UTRs ranging from 58 to 59 in length and 3'UTRs ranging from 268 to 323 nucleotides. A minimum of 11–12 residues in the poly(A) tail are required for efficient production of negative-strand RNA, negative-strand synthesis in combination with CSE, and for the poly(A) tail to function in supporting efficient translation.

[0473] An exemplary srRNA vector structure is shown in FIG. 15. The vector contains six parts: 5' cap, 5' untranslated region, viral nonstructural proteins (nsP1-4), subgenomic promoter, 3' untranslated region, and poly(A) tail. The 5' UTR controls translation, and the 5' end open reading frame (ORF) is translated into viral nonstructural proteins (nsP1-4). These proteins form an enzyme complex required for replication of the viral genome and transcription of the subgenomic RNA. The srRNA vector structure optimizes the type of cap, 3' untranslated region, and poly(A) tail to increase RNA stability (increase half-life) in vivo. The structure is based on naturally occurring alphaviruses, utilizing cap 1 and an optimized 3' UTR, making the vector resemble a real virus replicating in vivo. Furthermore, it has been observed that increasing the length of the poly(A) tail improves the efficiency of polysome generation, which in turn affects protein expression levels. The disclosed extended poly(A) tails are extended to 65 or 95 residues and promote srRNA stability and translation of the viral transcript.

[0474] Gene synthesis and recombinant plasmid cloning

[0475] DNA encoding the gene of interest (GOI) was chemically synthesized and cloned into a restriction-linearized pUC57 plasmid vector. DNA synthesis and gene cloning were customized and ordered from Nanjing Genscript Co., Ltd. (Nanjing, China). The srRNA replicon backbone was chemically synthesized by Suzhou Genwitz Co., Ltd. (Suzhou, China). The GOI and replicon backbone were digested with ApaI and NotI by T4 DNA ligase, and the GOI sequence was inserted into the backbone to obtain the recombinant plasmid. Sequencing proved that the GOI was accurately inserted into the expression backbone.

[0476] RNA synthesis

[0477] RNA was produced in vitro using T7 RNA polymerase-mediated transcription from a linearized DNA template derived from a recombinant plasmid encoding the codon-optimized RBD region of SARS-CoV-2, incorporating 5' and 3' untranslated regions and a polyA tail. RNA encoding the VZV gE protein was prepared by the same procedure.

[0478] cell line

[0479] HEK-293T and BHK-21 cells were purchased from the National Collection of Authenticated Cell Cultures (https: / / www.cellbank.org.cn / ).

[0480] Cell culture and transfection

[0481] HEK-293T and BHK-21 were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin / streptomycin antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin; Gibco) and maintained at 37°C, 5% CO2, and 90% relative humidity. 3 × 10 cells per well were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (Gibco) and penicillin / streptomycin antibiotics (100 U / mL penicillin, 100 μg / mL streptomycin; Gibco). 5 Cells were seeded in 6-well plates. The next day, when the cell culture should have a viability of more than 90% and reach 70% confluence, 2 μg of srRNA per well of 6-well plates was transfected with Lipofectamine 3000 Transfection Kit (Invitrogen, 2320817). Samples were taken 24 and 48 hours after transfection for the following detection.

[0482] In vitro potency assay

[0483] The RBD protein of SARS-CoV-2 was detected in the cell supernatant by Western blot. The RNA-transfected cells were lysed with NP-40 cell lysis buffer (50 mM Tris, pH 7.4, 150 nM NaCl, 1% NP40, sodium pyrophosphate, β-glycerophosphate, sodium orthovanadate, sodium fluoride, EDTA, leupeptin). The mixture was centrifuged at 13,000 rpm for 5 min at 4°C. The supernatant was collected, boiled with SDS for 12 min at 95°C, separated on a 6% SDS-PAGE gel, and transferred to a nitrocellulose filter membrane. After blocking with 5% BSA, the membrane was first blotted with primary antibody (1:1000) (SARS-CoV-2 (2019-nCoV) spiked rabbit PAb, 40592-T62, Sino Biological), then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:10000) (IgG(H+L) (HRP-labeled goat anti-rabbit IgG(H+L)), Beyotime) and visualized with chemiluminescence reagent (C chemiluminescence HRP substrate, WBKLS0500, Millipore). VZV gE protein was detected by the same method using anti-VZV gE protein antibody (abcam, ab272686).

[0484] Preparation of RNA-LNPs

[0485] Lipid nanoparticles (LNPs) were formulated using a microfluidic device by rapidly mixing the ethanol phase and the aqueous phase. The aqueous phase was 50 mM citrate buffer (pH 6.0) and contained a specific amount of RNA. The ethanol phase contained ionizable lipids (as described herein), cholesterol (Jiangsu Southeast Nanomaterials), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Jiangsu Southeast Nanomaterials), and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (SINOPEG). These four lipid components were mixed in a molar ratio of 40:48:10:2.0 (ionizable lipid:cholesterol:DSPC:DMG-PEG2000). The resulting LNPs were purified in citrate buffer (pH 6.0) and the particle size, polydispersity index (PDI), and RNA concentration were characterized. Finally, the pH was adjusted to 7.2. The RNA-LNP was then diluted to the target concentration to obtain the RNA-LNP product, which was stored at -80°C.

[0486] For characterization, the particle size and polydispersity index (PDI) of the RNA-LNPs were determined by dynamic light scattering (DLS).

[0487] Bioluminescence Imaging (BLI)

[0488] Six to eight week old female BALB / C mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Two micrograms of LNP-srRNA was administered intramuscularly to the mice. Before the test time point, the mice were anesthetized in a chamber containing 2.5% isoflurane and administered D-luciferin K+ salt XenoLight (Perkin Elmer, P / N 122799, 150 mg / kg) via intraperitoneal injection. Luminescence was detected with an IVIS Spectral Imaging System (PerkinElmer Lumina LT, Waltham, MA) while maintaining 2% isoflurane in the imaging chamber through a nose cone. Images were captured 10 minutes after luciferin administration with the autoexposure device set to sequence. Total photon flux values ​​(photons / sec) corresponding to regions of interest (ROIs) marked around the bioluminescent signal were analyzed using Caliper Life Sciences software (Living IMAGE Software, Caliper).

[0489] Antigen-specific antibody reaction

[0490] Blood was collected from the retro-orbital sinus of immunized mice and serum was prepared. Antigen-specific IgG responses were detected by enzyme-linked immunosorbent assay (ELISA) using commercial antigens: VZV gE protein (abcam ab43050) and SARS-CoV-2 spike protein (RBD, Histag) (GenScript Z03483). Briefly, 0.5 μg / mL of antigen diluted in carbonate-bicarbonate buffer was precoated on 96-well clear polystyrene microplates (Corning) overnight at 4°C. After washing three times with PBS-T (0.05% Tween-20 in PBS), the coated plates were blocked with 300 μL of blocking buffer (15% normal goat serum and 2% bovine serum albumin in PBS-T) for 1 h at 37°C. Serum samples were serially diluted 2-fold in blocking buffer, transferred to the plate and incubated for 1 hour at 37°C. After washing, the plate was incubated with HRP-conjugated rabbit anti-mouse IgG H+L (Abcam, ab6728) for 1 hour at 37°C. The plate was washed and incubated with TMB single component substrate solution (Solarbio, PR1200) for 7 minutes at 37°C, and the reaction was stopped with ELISA stop solution (Solarbio, C1058). Absorbance was read at 450 nm in a microplate reader (VARIOSKAN LUX, ThermoFisher) and ELISA titers were determined using nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software.

[0491] Intracellular cytokine staining and flow cytometry - cell stimulation

[0492] Fresh splenocytes (2 × 10 in 200 µL medium) 6) were seeded into 96-well round-bottom microplates and restimulated with 1.25 μg / mL of VZV gE protein (a pool of 138 15-mer peptides with an overlap of 11 amino acids) and 1.25 μg / mL of a pool of peptides spanning CD28 and CD49d monoclonal antibodies. Plates were incubated at 37° C. in a humidified incubator with 5% CO2 for 2 hours, followed by overnight treatment with a protein transport inhibitor cocktail.

[0493] Intracellular cytokine staining and flow cytometry - cell surface and intracellular staining

[0494] The cells were washed with Dulbecco's phosphate buffered saline (DPBS) and LIVE / DEAD TM Fixed aqueous dead cell staining (LIVE / DEAD TM The cells were stained with fixable aqua dead cell stain (FACS) for 30 minutes. The cells were washed with 200 μL of fluorescence-activated cell sorting (FACS) wash buffer and incubated with fluorescently labeled primary antibodies for 30 minutes to stain cell surface proteins. Antibodies included anti-mouse CD3 APC-Vio 770 (clone REA641), anti-mouse CD4 VioBlue (clone REA604), and anti-mouse CD8 PerCP (clone REA601) (1 μL per well). The cells were then washed with FACS wash buffer and incubated with fixative / permeabilization standard solution for 30 minutes according to the manufacturer's instructions. The cells were then washed twice with permeabilization buffer and incubated with fluorescently labeled primary antibodies for 30 minutes to detect cytokine expression in the cells. Antibodies included anti-mouse IFN-γ FITC (clone REA638), anti-mouse TNF-α PE (clone REA636), and anti-mouse IL-2 APC (clone REA665) (1 μL per well). Finally, cells were washed with 200 μL of permeabilization buffer and resuspended in 200 μL of PBS for flow cytometry analysis.

[0495] Stained cells were analyzed using a CytoFlex flow cytometer (Beckman). Data was analyzed using CytExpert software.

[0496] Construction and quality control of srRNA vaccines

[0497] The srRNA constructs used are shown in Figure 16. Three model GOIs were used to evaluate in vitro and in vivo potency. To evaluate in vitro potency, SARS-CoV RBD srRNA constructs were generated by in vitro transcription (IVT) followed by transfection of cells with Lipofectamine 3000 followed by RBD protein expression analysis by WB. To evaluate in vivo potency, VZV gE and luciferase srRNA constructs were produced by in vitro transcription (IVT) and formulated in LNPs containing ionizable lipid (lipid #9), cholesterol, DSPC, and DMG-PEG2000. For all in vivo experiments, the size, polydispersity index (PDI), encapsulation efficiency, and concentration of the LNPs were characterized (Table 14). As shown in Table 14, the characterization of RNA-LNPs for srRNA of constructs 1, 3-6 are all suitable for delivering RNA. [Table 14]

[0498] RNA in vitro transfection

[0499] The transfection efficiency of RBD RNA in the four constructs was compared with the HEK-293T and BHK-21 cell lines at the same dose (1 μg). In this study, RBD protein was detected in the cell supernatant 24 hours after transfection using Western blot, as shown in Figures 17A and 17B. Based on the results shown in Figures 17A and 17B, it can be concluded that the four constructs disclosed herein are capable of expressing RBD protein. Comparing the results of Structure 1 and Structure 2 in HEK-293T and BHK-21, the expression of RBD in Structure 2 is lower than Structure 1. Comparing the results of Structure 2 and Structure 3 in HEK-293T and BHK-21 cells, the expression of RBD in Structure 3 is lower than Structure 2. Thus, the data indicates that increasing the length of the polyA tail can increase the stability of the RNA while slowing down the initial translation efficiency. Minimization of the 3'UTR to 3'UTR 330 can also slow down the initial translation efficiency and extend the time of in vivo expression.

[0500] Efficacy of RNA-LNP vaccines in mice

[0501] The in vitro expression results show that the optimized constructs can reduce the initial expression amount. The next step is to verify whether the optimized constructs can extend the RNA expression cycle in animals. In vivo studies were performed to compare the protein expression cycle in BALB / c mice upon immunization with luciferase RNA-LNPs encoding luciferase protein. The in vivo expression of GOI in different constructs in mice was evaluated. To visualize the expression of RNA-LNP vaccine, luciferase encoding RNA-LNPs were prepared and subjected to bioluminescence imaging (BLI) using intramuscular injection (im). After im, robust expression of luciferase was confirmed at the injection site of BALB / c mice 5 days after injection, and the length of expression time of constructs 3 and 4 was longer than that in construct 1, as shown in Figures 18 and 19. Specifically, luciferase expression is detectable between days 5 and 9 for construct 1, and between days 5 and 21 for constructs 3 and 4. Optimized Structures 3 and 4, with 3'UTR 330 and extended polyA tails, can increase the in vivo half-life of the RNA.

[0502] In vivo immunogenicity evaluation of srRNA-VZV vaccine in C57BL / 6 mice

[0503] The in vivo luciferase protein expression results confirm that the disclosed srRNA structures with 3'UTR 330 and long polyA tails can express proteins for longer periods than srRNA structures without these features. The next step was to compare the antibody levels produced by the expressed antigen proteins. Here, VZV gE proteins of Structure 5, Structure 6, and Structure 7 were expressed, and the durability of specific antibody responses in Bkack6 / C57 mice upon immunization with VZVgE RNA-LNP was compared. The srRNAs encoding VZVgE proteins in Structure 5, Structure 6, and Structure 7 use Cap 1 as Cap 1. These three srRNA-VZV vaccines used Structure 5, Structure 6, and Structure 7, as shown in FIG. 16.

[0504] To mimic natural VZV infection and subsequent latency, mice were pre-immunized subcutaneously (sc) with varicella-zoster virus, live (LAV) vaccine (3.3 μg plaque-forming units (PFU)) in the nape of the neck 35 days (day -35) prior to the start of the study. Mice were immunized twice with 2 μg VZV vaccine per dose administered intramuscularly (im) at an interval of 42 days, as shown in Figure 20A, which is a schematic diagram of the VZVgE RNA-LNP immunization process in mice.

[0505] Antibody titers are highly correlated with the protective effect and durability provided by the vaccine, and therefore are used as efficacy readouts in this study, presented in the form of total IgG endpoint titers. As shown in Figure 20B, after the first injection, the vaccine of Structure 6 induced the maximum antibody response at 14, 35, and 42 days after the first immunization. For all three RNA vaccines, the antibody titers increased significantly after two immunizations. However, after two immunizations, the vaccines of Structure 6 and Structure 7 seemed to induce higher or equal antibody responses for a long time, e.g., more than 90 days after the first administration. These data demonstrate that the RNA-LNP vaccines of Structure 6 and Structure 7 are capable of producing persistent and specific antibodies in the body. Considering the stability of plasmid passage and the difficulty of gene synthesis for longer RNAs, Structure 6 is more suitable for large-scale production. Both Structures 6 and 7 are suitable for sustained srRNA expression of GOI and improved immunogenicity.

[0506] To measure the frequency and magnitude of antigen-specific T cell subsets, PBMCs of immunized mice were stimulated with peptides spanning the VZV gE protein to test their response capacity by producing T cells expressing IFN-γ and / or TNF-α. On day 57, some mice in each group were harvested and splenocytes were collected for flow cytometry to measure cellular immune responses. As shown in Figure 21A, the frequency of total cytokine (IFN-γ and / or TNF-α) expressing GD4+T in Structure 6 was higher than that in Structure 5 and Structure 7. Thus, Structure 6 was able to induce a significant VZV gE-specific CD4+T cell immune response. As shown in Figure 21B, the frequency of total cytokine (IFN-γ and / or TNF-α) expressing GD8+T in Structure 6 was higher than that in Structure 5 and Structure 7. Thus, Structure 6 was also able to induce a significant VZV gE-specific CD8+T cell immune response. The results showed that the vaccine prepared with the novel self-replicating vector construct 6 was able to induce the strongest immune response.

[0507] Example 24. Synthesis of lipid #4 [ka] [ka]

[0508] Step 1. Synthesis of compound 2

[0509] To a solution of compound 1 (60.0 g, 298 mmol) in DMF (420 mL), K2CO3 (82.5 g, 596 mmol) was added, and the mixture was stirred at 25 °C for 10 min, after which compound 6a (104 g, 298 mmol) was added, and the mixture was stirred at 80 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, product Rf = 0.6) showed that compound 1 was completely consumed and the desired spot was detected. The reaction mixture was filtered, extracted with EtOAc (200 mL x 2), washed with 100 mL of brine, dried over Na2SO4, and concentrated under reduced pressure to give a residue. Compound 2 (95.0 g, crude) was obtained as a brown oil.

[0510] Step 2. Synthesis of compound 3

[0511] To a solution of compound 2 (95.0 g, 202 mmol) in MeOH (475 mL) and THF (285 mL), NaBH4 (3.06 g, 80.9 mmol) was added at 0° C., and the mixture was stirred at 25° C. for 2 h. TLC (petroleum ether / ethyl acetate=10 / 1, product Rf=0.5) showed that compound 2 was completely consumed and the desired spot was detected. The reaction mixture was quenched with 200 mL of MeOH, extracted with EtOAc (200 mL×2), washed with 100 mL of brine, dried over Na2SO4, and concentrated under reduced pressure to give a residue. Compound 3 (67.0 g, crude) was obtained as a brown oil.

[0512] Step 3. Synthesis of compound 4

[0513] To a solution of compound 3 (67.0 g, 142 mmol) in DMF (420 mL), imidazole (14.1 g, 213 mmol), tert-butyl-chloro-diphenyl-silane (46.7 g, 170 mmol) were added at 0 °C, and the mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, product Rf = 0.7) showed that compound 3 was completely consumed and the desired spot was detected. H2O (300 mL) was added, and the reaction mixture was extracted with DCM (400 mL × 2), washed with 100 mL of brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 1 / 0 to 0 / 1). Compound 4 (98.0 g, 117 mmol, 82.5% yield, 85.0% TLC purity) was obtained as a yellow oil. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 1 / 0 to 0 / 1). Compound 4 (98.0 g, 117 mmol, yield 82.5%, purity 85.0%) was obtained as a yellow oil.

[0514] Step 4. Synthesis of compound 5

[0515] To a solution of compound 4 (98.0 g, 138 mmol) in dioxane (630 mL), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (52.5 g, 207 mmol), Pd(dppf)Cl2 (20.2 g, 27.6 mmol), KOAc (27.1 g, 276 mmol) were added and the mixture was stirred at 75 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, product Rf = 0.5) showed that compound 4 was completely consumed and the desired spot was detected. The reaction mixture was filtered, extracted with DCM (300 mL x 2), washed with 100 mL of brine, dried over Na2SO4, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane=1 / 0-10 / 1). Compound 5 (62.5 g, 82.5 mmol, 59.8% yield) was obtained as a brown oil.

[0516] Step 5. Synthesis of compound 6

[0517] A solution of compound 5, H2O2 (18.7 g, 165 mmol, purity 30.0%), and NaOH (1 M, 82.5 mL) in THF (420 mL) was stirred at 25 °C for 2 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf of product = 0.4) showed that compound 5 was completely consumed and the desired spot was detected. The reaction mixture was quenched by adding Na2SO3 (250 mL aq.) at 0 °C, and then extracted with EtOAc (200 mL × 2). The combined organic layers were washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 1 / 0 to 5 / 1). Compound 6 (37.0 g, 57.1 mmol, yield 69.2%) was obtained as a yellow oil.

[0518] Step 6. Synthesis of compound 7

[0519] To a solution of compound 6 (37.0 g, 57.1 mmol) in DMF (200 mL), K2CO3 (15.8 g, 114 mmol), compound 1a (26.4 g, 57.1 mmol) were added, and the mixture was stirred at 80 °C for 5 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf of product = 0.6) showed that compound 6 was completely consumed and the desired spot was detected. 500 mL of H2O was added, and the reaction mixture was extracted with DCM (200 mL x 2). The combined organic layers were washed with aqueous LiCl solution (100 mL x 2), washed with brine (100 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 1 / 0 to 0 / 1). Compound 7 (41.0 g, 39.9 mmol, 69.7% yield) was obtained as a yellow oil.

[0520] Step 7. Synthesis of compound 8

[0521] To a solution of compound 7 (41.0 g, 39.9 mmol) in THF (280 mL), TBAF (1 M, 79.8 mL) was added and the mixture was stirred at 25 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, Rf = 0.50) showed that compound 7 was consumed and the desired spot was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 1 / 0 to 3 / 1). Compound 8 (20.5 g, 25.9 mmol, 65.1% yield) was obtained as a yellow oil.

[0522] Step 8. Synthesis of the compound dinonyl 8,8'-((2-(((4-(dimethylamino)butanoyl)oxy)methyl)-1,4-phenylene)bis(oxy))dioctanoate

[0523] To a solution of 4-(dimethylamino)butyric acid (5.20 g, 39.6 mmol) in Py (20 mL), EDCI (10.1 g, 52.8 mmol) and compound 8 (20.5 g, 26.5 mmol) were added, and the mixture was stirred at 50 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, product Rf = 0.01) showed that compound 8 was completely consumed and the desired spot was detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / dichloromethane = 10 / 0 to 0 / 1). Lipid #4 (11.9 g, 13.3 mmol, 50.3% yield, 99.4% purity) was obtained as a pale yellow oil.

[0524] Example 25. Synthesis of lipid #5 [ka] [ka]

[0525] Step 1. Synthesis of compound 2

[0526] To a solution of compound 1 (25.0 g, 124 mmol) in DMF (175 mL) was added compound 1a (47.7 g, 136 mmol) and K2CO3 (34.3 g, 248 mmol). The mixture was stirred at 80° C. for 12 h. TLC (petroleum ether / ethyl acetate=10 / 1, R f =0.5) indicated that compound 1 was completely consumed and the desired spot was detected. The reaction mixture was diluted with 300 mL of H2O and 20.0 mL of THF, and then extracted with EtOAc (200 mL x 3). The combined organic layers were washed with 50.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. Compound 2 (57.0 g, crude) was obtained as a brown oil.

[0527] Step 2. Synthesis of compound 3

[0528] A solution of compound 2 (20.0 g, 34.38 mmol) was dissolved in THF (40.0 mL) and MeOH (100 mL), then the mixture was cooled to 0° C. and NaBH4 (520 mg, 13.7 mmol) was added. The reaction was stirred at 25° C. for 3 h. TLC (petroleum ether / ethyl acetate=10 / 1, R of product f =0.3) indicated that compound 2 was completely consumed and the desired spot was detected. The reaction mixture was quenched with 20.0 mL of aqueous NH4Cl and extracted with EtOAc (50.0 mL x 3). The combined organic layers were washed with 10.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. Compound 3 (20.0 g, crude) was obtained as a brown oil.

[0529] Step 3. Synthesis of compound 4

[0530] A solution of compound 2 (20.0 g, 34.38 mmol) was dissolved in THF (40.0 mL) and MeOH (100 mL), then the mixture was cooled to 0° C. and NaBH4 (520 mg, 13.7 mmol) was added. The reaction was stirred at 25° C. for 3 h. TLC (petroleum ether / ethyl acetate=10 / 1, R of product f=0.3) indicated that compound 2 was completely consumed and the desired spot was detected. The reaction mixture was quenched with 20.0 mL of aqueous NH4Cl and extracted with EtOAc (50.0 mL x 3). The combined organic layers were washed with 10.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. Compound 3 (20.0 g, crude) was obtained as a brown oil.

[0531] Step 4. Synthesis of compound 5

[0532] A mixture of compound 4 (10.0 g, 12.1 mmol), KOAc (2.39 g, 24.3 mmol), 4,4,5,5-tetramethyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,3,2-dioxaborolane (3.71 g, 14.6 mmol), Pd(dppf)Cl2 (890 mg, 1.22 mmol) in 1,4-dioxane (70.0 mL) was degassed and purged with N2 three times, after which the mixture was stirred at 75 °C under N2 atmosphere for 12 h. TLC (petroleum ether:ethyl acetate = 10 / 1, R f =0.7) indicated the reaction was complete. The reaction was filtered and concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 5 / 1). Compound 5 (13.0 g, crude) was obtained as a yellow oil.

[0533] Step 5. Synthesis of compound 6

[0534] To a solution of compound 5 (13.0 g, 14.9 mmol) in THF (130 mL), HO (3.39 g, 29.9 mmol, 30% purity) and NaOH (1 M, 14.9 mL) were added and stirred at 25° C. for 1 h. TLC (petroleum ether / ethyl acetate=10 / 1, R f=0.43) indicated the reaction was complete. The reaction mixture was quenched by adding 100 mL of Na2SO3 at 0 °C, and then extracted with EtOAc (100 mL × 2). The combined organic layers were washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 1 / 1). Compound 6 (10.0 g, crude) was obtained as a yellow oil.

[0535] Step 6. Synthesis of compound 7

[0536] To a solution of compound 6 (9.50 g, 12.5 mmol) and nonyl 8-bromooctanoate (5.03 g, 14.4 mmol) in DMF (63.0 mL), K2CO3 (3.46 g, 25.0 mmol) was added and stirred at 90 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1, R f =0.7) indicated the reaction was complete. The reaction mixture was extracted with solvent (100 mL × 3). The combined organic layers were washed with 100 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 1 / 1). Compound 7 (6.30 g, 6.13 mmol, 48.9% yield) was obtained as a yellow oil.

[0537] Step 7. Synthesis of compound 8

[0538] To a solution of compound 7 (6.30 g, 6.13 mmol) in THF (30.0 mL), TBAF (1 M, 12.2 mL) was added and stirred at 25° C. for 5 h. TLC (petroleum ether / ethyl acetate=10 / 1, R f=0.3) indicated the reaction was complete. The reaction mixture was extracted with EtOAc (50.0 mL×2). The combined organic layers were washed with 50.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=50 / 1 to 1 / 1). Compound 8 (6.00 g, crude) was obtained as a yellow oil.

[0539] Step 8. Synthesis of lipid #5

[0540] To a solution of compound 8 (6.00 g, 7.60 mmol), 4-(dimethylamino)butyric acid (1.91 g, 11.4 mmol, HCl salt) in pyridine (6.00 mL), EDCI (2.91 g, 15.2 mmol) was added at 25° C. and stirred at 50° C. for 12 hours. TLC (petroleum ether / ethyl acetate=1 / 1, R f =0.1) indicated the reaction was complete. The reaction was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1). Lipid #5 (3.00 g, 3.32 mmol, 43.7% yield, 100% purity) was obtained as a yellow oil.

[0541] Example 26. Synthesis of lipid #8 [ka]

[0542] Step 1. Synthesis of compound 2

[0543] To a solution of compound 1 (4.92 g, 14.0 mmol) in DMF (17.0 mL) was added K2CO3 (4.01 g, 27.8 mmol) and 2,5-dihydroxybenzaldehyde (1.01 g, 7.0 mmol). The mixture was stirred at 90 °C for 12 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 1 was completely consumed. The crude reaction mixture was poured into water (10.0 mL) and extracted with ethyl acetate (10.0 mL x 2). The combined organic layers were washed with 10.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 1 to 50 / 1). Compound 2 (3.99 g, 4.06 mmol, 58.0% yield) was obtained as a brown oil.

[0544] Step 2. Synthesis of compound 3

[0545] A solution of compound 2 (3.99 g, 4.06 mmol) was dissolved in THF (20.0 mL) and MeOH (100 mL), then the mixture was cooled to 0 °C and NaBH4 (520 mg, 13.7 mmol) was added. The reaction was stirred at 25 °C for 3 h. TLC (petroleum ether / ethyl acetate = 10 / 1) showed that compound 2 was completely consumed and the desired spot was detected. The reaction mixture was quenched with 20.0 mL of aqueous NH4Cl solution and extracted with EtOAc (50.0 mL × 3). The combined organic layers were washed with 10.0 mL of brine, dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 10 / 1). Compound 3 (3.56 g, 3.61 mol, 89.0% yield) was obtained as a brown oil.

[0546] Step 3. Synthesis of lipid #8

[0547] To a solution of compound 3 (3.56 g, 3.61 mmol), 4-(dimethylamino)butyric acid (0.95 g, 5.41 mmol, HCl salt) in pyridine (6.00 mL), EDCI (1.46 g, 7.2 mmol) was added at 25 °C and stirred at 50 °C for 12 h. TLC (petroleum ether / ethyl acetate = 1 / 1) showed the reaction was complete. The reaction was concentrated under reduced pressure. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10 / 1 to 0 / 1). Lipid #8 (2.42 g, 2.20 mmol, 61% yield) was obtained as a pale yellow oil.

[0548] Example 27. Synthesis of lipid #9 [ka] [ka]

[0549] Step 1: Synthesis of Compound 1-2

[0550] Compound 1-1 (100 g, 495 mmol, 90.1 mL) was added to a solution of NaOEt (33.7 g, 495 mmol) in EtOH (250 mL). With stirring, the solution was heated to 80° C. and compound 1-1A (124 g, 495 mmol) was added dropwise to the solution. The solution was stirred at 80° C. for 2 h. Another solution of NaOEt (33.7 g, 495 mmol) in EtOH (250 mL) was added to the reaction solution, followed by compound 1-1A (124 g, 495 mmol). The solution was stirred at 80° C. for 12 h. LCMS showed the reaction was complete. The reaction suspension was concentrated under vacuum to give a residue. The residue was added to H2O (100 mL) and the pH of the solution was adjusted to 7 with aqueous HCl (0.5 M). The solution was extracted with MTBE (150 mL x 2) and the organic layer was washed with brine (100 mL x 2). The combined organic layers were concentrated under vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 250 / 1 to 1 / 10) to give compound 1-2 (166 g, 306 mmol, yield 61.9%) as a light brown oil.

[0551] Step 2 Synthesis of compounds 1-3

[0552] To a solution of compound 1-2 (165 g, 304 mmol) in THF (450 mL) and MeOH (250 mL) was added an aqueous solution of LiOH.H2O (4.0 M, 456 mL). The solution was stirred at 20 °C for 5 h. LCMS showed the reaction was complete. The reaction was concentrated under vacuum to give the intermediate product (150 g, crude) as a yellow solid, which was used directly in the next step. A solution of the intermediate product (15.0 g, 34.8 mmol) in HCl (12.0 M, 53.5 mL) and AcOH (80.0 mL) was stirred at 70 °C for 12 h. LCMS showed the reaction was complete. The suspension was concentrated under reduced pressure to give a suspension. The pH of the suspension was adjusted to 2 with HCl (12.0 M). The suspension was then filtered and the filter cake was concentrated under reduced pressure to give compound 1-3 (9.50 g, 27.7 mmol, 79.6% yield) as a brown solid, which was used directly in the next step.

[0553] Step 3 Synthesis of compound 1-3-1

[0554] To a solution of compound 1-3 (40.0 g, 116 mmol) in DCM (250 mL) was added DMF (85.3 mg, 1.17 mmol, 89.8 μL) and (COCl)2 (30.4 g, 239 mmol, 20.9 mL). The solution was stirred at 20° C. for 3 h. A sample was taken and quenched with MeOH. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.68) indicated that the starting material was completely consumed. The reaction solution was concentrated under vacuum to give a residue. The residue was dissolved in toluene (30.0 mL x 2) and concentrated under vacuum twice to give compound 1-3-1 (44.0 g, crude) as a dark brown oil, which was used directly in the next step.

[0555] Step 4 Synthesis of compounds 1-4

[0556] To a solution of compound 1-3A (34.6 g, 243 mmol) in DCM (160 mL) was added TEA (35.2 g, 347 mmol, 48.4 mL). A solution of compound 1-3-1 (44.0 g, 115 mmol) in DCM (60.0 mL) was added dropwise to the solution. The solution was stirred at 20° C. for 12 hours. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.79) indicated that the starting material was completely consumed. The reaction solution was poured into H2O (250 mL) and the suspension was extracted with ethyl acetate (150 mL × 2). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 500 / 0-0 / 1) to give compound 1-4 (21.0 g, 35.5 mmol, yield 30.6%) as a brown solid.

[0557] Step 5 Synthesis of compounds 1-5

[0558] To a solution of compound 1-4 (21.0 g, 35.5 mmol) in THF (80.0 mL) and H2O (20.0 mL) was added NaBH4 (26.9 g, 710 mmol). The solution was stirred at 0 °C for 1 h. TLC (petroleum ether: ethyl acetate = 3:1, R of the product f =0.74) indicated the reaction was completed. The reaction mixture was poured into aqueous NH4Cl (50.0 mL), and the solution was extracted with ethyl acetate (10.0 mL x 2). The organic layer was extracted with H2O (10.0 mL x 2). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 200 / 0-0 / 1) to give compound 1-5 (9.00 g, 15.2 mmol, 42.7% yield) as an off-white oil.

[0559] Step 6 Synthesis of lipid #9

[0560] To a solution of compound 1-5 (14.0 g, 23.6 mmol) in Py (60.0 mL), compound 1-5A (7.92 g, 47.2 mmol) was added. EDCI (9.05 g, 47.2 mmol) was added to the solution. The solution was stirred at 45 °C for 2 h. LCMS showed the reaction was complete. The reaction mixture was poured into aqueous NH4Cl (80.0 mL) and the solution was extracted with ethyl acetate (30.0 mL x 2). The organic layer was washed with H2O (30.0 mL x 2). The organic layer was dried over sodium sulfate and concentrated under reduced pressure to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 0 to 0 / 1) to obtain lipid #9 (15.0 g, 21.2 mmol, 90% yield) as a pale yellow oil.

[0561] Example 28. Synthesis of lipid #11 [ka] [ka] [ka]

[0562] Step 1: Synthesis of Compound 1-3C2

[0563] NaH (24.0 g, 599 mmol, 60% purity, 0.36 equiv) was added dropwise to a solution of 1-3C1 (150 g, 1.66 mol, 147 mL, 1 equiv) in DMF (1500 mL) at -10 °C. After 30 min, 1-bromohexane (98.9 g, 599 mmol, 83.8 mL, 0.36 equiv) was added to the solution. The solution was stirred at 15 °C for 3 h. TLC (petroleum ether:ethyl acetate=1:1, R of the product) showed no significant difference from the control. f=0.40) indicated the reaction was completed. The solution was poured into H2O (2 L). The aqueous phase was extracted with EtOAc (1 L × 3). The combined organic layers were washed with brine (2 L), and the resulting organic layer was dried over Na2SO4 and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10:1 to 1:10) to give compound 1-3C2 (60.0 g, 344 mmol, 20.7% yield) as a yellow oil.

[0564] Step 2 Synthesis of compounds 1-3C3

[0565] (COCl)2 (43.9 g, 346 mmol, 30.3 mL, 1.30 equiv) in DCM (1500 mL) was added to DMSO (33.3 g, 426 mmol, 33.3 mL, 1.60 equiv) at -65°C. After 30 min, a solution of compound 1-3C2 (58 g, 266 mmol, 80% purity, 1 equiv) was added to the mixture, and after 30 min, TEA (121 g, 1.20 mol, 167 mL, 4.50 equiv) was added. The solution was stirred at -65°C for 30 min and at 15°C for another 30 min. TLC (petroleum ether:ethyl acetate=2:1, R of product f =0.50) indicated the reaction was completed. The solution was poured into H2O (500 mL). The above solution was extracted with DCM (500 mL × 2). The combined organic layers were dried in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 30:1 to 1:10) to give 1-3C3 (80 g, 245 mmol, yield 54.0%) as a colorless oil.

[0566] Step 3 Synthesis of compounds 1-3C4

[0567] A solution of compound 1-3C3 (38.0 g, 221 mmol, 1 equiv) and ethyl 2-(triphenylphosphoranylidene)acetate (84.5 g, 243 mmol, 1.1 equiv) in DCM (200 mL) was stirred at 15 °C for 6 h. TLC (petroleum ether:ethyl acetate=5:1, product Rf=0.60) and LCMS showed the reaction was complete. The reaction solution was dried in vacuum to give a residue. The residue was triturated with petroleum ether (300 mL) at 15 °C for 30 min. The above suspension was filtered and the filtrate was dried in vacuum to give the crude product. The crude product was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=100:1 to 30:1) to give 1-3C4 (40 g, 165 mmol, 74.8% yield) as a yellow oil.

[0568] Step 4 Synthesis of compounds 1-3C

[0569] A solution of compound 1-3C4 (17.6 g, 72.6 mmol, 1 equiv) in DCM (50 mL) was cooled to -65°C. DIBAl-H (1 M, 152.5 mL, 2.1 equiv) was added dropwise to the above solution. The solution was stirred at -65°C for 2 h. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.20) indicated the reaction was completed. The reaction solution was added dropwise to glacial HCl (4M, 100 mL) to adjust the pH of the solution to 2. Then, the solution was extracted with DCM (300×3), and the combined organic layers were dried over Na2SO4 and dried in vacuum to obtain the crude product. The crude product was added dropwise to glacial aqueous HCl (4M, 100 mL) until the pH value reached 2. Then, the solution was extracted with DCM (300×3), and the combined organic layers were dried over Na2SO4 and dried in vacuum to obtain 1-3C (12 g, 59.91 mmol, 82.49% yield) as a yellow oil.

[0570] Step 5 Synthesis of compounds 1-5

[0571] To a solution of DCC (7.95 g, 38.5 mmol, 7.79 mL, 2.1 equiv) and 1-3 (6.28 g, 18.3 mmol, 1 equiv) in DCM (50 mL) was added 1-3C (7.71 g, 38.5 mmol, 2.1 equiv) and DMAP (1.12 g, 9.17 mmol, 0.5 equiv). The solution was stirred at 15 °C for 2 h. TLC (petroleum ether:ethyl acetate = 5:1, R of product f =0.60) indicated the reaction was completed. The reaction suspension was filtered and the filter cake was washed with DCM (50 mL × 2). The filtrate was washed with HCl (2 M, 30 mL) and the combined organic layer was dried in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 80:1 to 20:1) to give compound 1-5 (8 g, 8.49 mmol, yield 46.3%) as a white solid, which was used directly in the next step.

[0572] Step 6 Synthesis of compounds 1-6

[0573] A solution of compound 1-5 (5 g, 7.1 mmol, 1.0 equiv) in THF (30 mL) and EtOH (10 mL) was cooled to 0° C. NaBH4 (0.96 g, 25.4 mmol, 3.59 equiv) was added to the above solution. The solution was stirred at 0° C. for 5 h. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.30) indicated the reaction was complete. The solution was poured into ice H2O (100 mL) and the pH of the solution was adjusted to 2 with ice HCl (1 M). The above solution was extracted with EtOAc (100 mL × 3). The combined organic layers were dried over Na2SO4 and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100:1 to 5:1) to give compound 1-6 (4 g, 5.64 mmol, yield 79.8%) as a colorless oil.

[0574] Step 7 Synthesis of lipid #11

[0575] To a solution of compound 1-6 (4 g, 5.64 mmol, 1 equiv.) and 4-(dimethylamino)butyric acid (1.23 g, 7.33 mmol, 1.30 equiv., HCl) in pyridine (30 mL) was added EDCI (2.70 g, 14.1 mmol, 2.5 equiv.). The solution was stirred at 40° C. for 6 h. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.40) indicated the reaction was complete. The solution was poured into H2O (100 mL). The resulting solution was extracted with EtOAc (100 mL × 3). The combined organic layers were washed with brine (200 mL) and concentrated in vacuo to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 10:1 to 10:1) to give lipid #11 (1.22 mmol, 22% yield) as a yellow oil.

[0576] Example 29. Synthesis of lipid #12 [ka] [ka]

[0577] Step 1. Synthesis of compounds 1-9

[0578] To a solution of compound 1-3 (26.0 g, 75.9 mmol) in DCM (200 mL) was added compound SM1 (16.6 g, 64.5 mmol), DCC (23.5 g, 114 mmol, 23.0 mL), and DMAP (2.78 g, 22.8 mmol). The solution was stirred at 25° C. for 12 h. TLC (petroleum ether:ethyl acetate=3:1, R f =0.50) indicated the reaction was completed. The solution was poured into H2O (300 mL). Then, the solution was extracted with DCM (300 mL × 3). The combined organic layers were washed with H2O (100 mL). The organic layers were dried in vacuum to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 20 / 1 to 5 / 1) to obtain compound 1-9 (15.0 g, 25.8 mmol, yield 34.0%) as a yellow oil.

[0579] Step 2 Synthesis of compounds 1-11

[0580] To a solution of compound 1-9 (11.5 g, 19.8 mmol) and DCC (5.72 g, 27.7 mmol, 5.61 mL) in DCM (60 mL) was added DMAP (725 mg, 5.94 mmol). The solution was stirred at 15° C. for 30 min. Then, compound SM3-4 (3.89 g, 19.40 mmol) was added to the above solution. The solution was stirred at 15° C. for 2 h. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.60) indicated that the starting material was completely consumed. The solution was poured into H2O (100 mL). Then, the solution was extracted with MTBE (50 mL × 3). The combined organic layers were washed with brine (30 mL), and the resulting organic layer was dried in vacuum to obtain a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 80 / 1 to 30 / 1) to obtain compound 1-11 (12.0 g, 15.7 mmol, yield 79.4%) as a colorless oil.

[0581] Step 3 Synthesis of compounds 1-12

[0582] A solution of compound 1-11 (10.5 g, 13.8 mmol) in THF (50 mL) and EtOH (10 mL) was cooled to 0° C. NaBH4 (1.00 g, 26.4 mmol) was added to the above solution. The solution was stirred at 0° C. for 5 h. TLC (petroleum ether:ethyl acetate=5:1, R of product f =0.30) indicated the reaction was completed. The solution was poured into ice H2O (100 mL) and the pH of the solution was adjusted to 2 with ice HCl (1M). The above solution was extracted with EtOAc (100 mL × 3). The combined organic layers were dried over Na2SO4 and concentrated in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 100 / 1 to 5 / 1) to give compound 1-12 (9.00 g, 11.8 mmol, yield 85.5%) as a colorless oil.

[0583] Step 4 Synthesis of lipid #12 To a solution of compound 1-12 (6.00 g, 7.84 mmol) and compound SM (1.97 g, 11.8 mmol) in Py (30 mL) was added EDCI (3.76 g, 19.6 mmol). The solution was stirred at 35° C. for 3 hours. TLC (petroleum ether:ethyl acetate=1:1, R f =0.30) indicated the reaction was completed. The solution was poured into H2O (50 mL). The above solution was extracted with EtOAc (50 mL × 3). The combined organic layers were dried in vacuum to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate = 50 / 1 to 1 / 50) to give lipid #12 (1.10 g, 1.25 mmol, 16.0% yield) as a yellow oil.

[0584] Incorporation by Reference The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.

[0585] Equivalent

[0586] The present disclosure may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the present disclosure described herein. The scope of the present disclosure is therefore indicated by the appended claims, rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced within the scope of the present invention.

[0587] Sequence Listing [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4]

Table 15-5

Table 15-6

Table 15-7

Table 15-8

Table 15-9

Table 15-10

Table 15-11

Table 15-12

Table 15-13

Table 15-14

Table 15-15

[0588] References 1.Moffat J, Mo C, Cheng JJ,Sommer M,Zerboni L,Stamatis S,Arvin AM.Functions of the C-terminal domain of varicella-zoster virus glycoprotein E in viral replication in vitro and skin and T-cell tropism in vivo.J Virol.2004 Nov;78(22):12406-15.doi:10.1128 / sJVI.78.22.12406-12415.2004.PMID:15507627;PMCID:PMC525039.

Claims

1. A kit comprising, separately, (1) a lyophilized polynucleotide composition and (2) a liquid lipid nanoparticle (LNP) solution, The LNP is (i) Formula II: 【Chemistry 1】 [In the formula, R 1 and R 2 are each independently C 1 -C 6 alkyl; R 3 is C 1 -C 5 alkyl; R 4 and R 5 are each independently a C 1 -C 18 alkyl group; Q 1 and Q 2 are each independently —O—C(O)—, —C(O)—O—, —O—C(S)—, —C(S)—O—, or —S—S—; and R 6 and R 7 are each independently C 1 -C 32 alkyl. or (ii) Formula I: 【Chemistry 2】 [In the formula, R 1 and R 2 are each independently C 1 -C 6 alkyl; R 3 is C 1 -C 5 alkyl; Q 1 , Q 2 , and Q 3 are each independently —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; L is C 1 -C 3 alkyl; R 4 and R 5 are each independently C 1 -C 10 alkyl; R 6 and R 7 are each independently C 1 -C 10 alkyl or C 1 -C 10 alkenyl; A 1 and A 2 are each independently a bond, —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; and R 8 and R 9 are each independently C 1 -C 30 alkyl. ionized lipids.

2. The ionizable lipid is 【Transformation 3】 The kit of claim 1, wherein

3. The kit of claim 1 , wherein the lyophilized polynucleotide composition comprises self-replicating RNA or encodes a protein, polypeptide, or antigen.

4. The ionizable lipid is 【Chemistry 4】 、 【Transformation 5】 、 【Transformation 6】 、 【Transformation 7】 、 【Transformation 8】 、 【Chemistry 9】 , and 【Chemistry 10】 2. The kit of claim 1, wherein the kit is selected from the group consisting of:

5. In the order 5' to 3', a) Cap1 cap; b) 5′UTR; c) one or more structural genes; d) gene of interest (GOI); e) a 3'UTR comprising about 100 to about 400 nucleotides; and f) a poly-A tail comprising about 30 to about 100 nucleotides A self-replicating RNA (srRNA) vector comprising:

6. 6. The srRNA vector of claim 5, wherein the 3'UTR comprises a nucleic acid sequence having at least about 80% identity to the nucleic acid sequence SEQ ID NO:

6.

7. 6. The srRNA vector of claim 5, wherein the GOI is a varicella-zoster virus (VZV) antigen, and the VZV antigen comprises a VZV glycoprotein E (gE) antigen.

8. The srRNA vector of claim 7 , wherein the sequence of the VZV antigen comprises SEQ ID NO: 2 or SEQ ID NO:

4.

9. The srRNA vector of claim 7, wherein the srRNA comprises a Cap1 cap, a VZV glycoprotein E (gE) antigen, a 3'UTR comprising approximately 270 nucleotides in length, and a polyA tail comprising approximately 65 nucleotides in length.

10. A pharmaceutical composition for preventing or treating herpes zoster or VZV-related diseases, comprising the kit of claim 1 or the srRNA vector of claim 5.

11. 1. A liquid composition comprising lyophilized RNA electrostatically adsorbed to or encapsulated in at least one outer portion of a lipid nanoparticle (LNP) comprising ionizable lipids at an ionizable lipid:RNA (N / P) molar ratio ranging from 5:1 to 12:1, The LNP is (i) Formula I: 【Chemistry 11】 [In the formula, R 1 and R 2 are each independently C 1 -C 6 alkyl; R 3 is C 1 -C 5 alkyl; Q 1 , Q 2 , and Q 3 are each independently —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; L is C 1 -C 3 alkyl; R 4 and R 5 are each independently C 1 -C 10 alkyl; R 6 and R 7 are each independently C 1 -C 10 alkyl or C 1 -C 10 alkenyl; A 1 and A 2 are each independently a bond, —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; and R 8 and R 9 are each independently C 1 -C 30 alkyl. or (ii) Formula II: 【Chemistry 12】 [In the formula, R 1 and R 2 are each independently C 1 -C 6 alkyl; R 3 is C 1 -C 5 alkyl; R 4 and R 5 are each independently a C 1 -C 18 alkyl group; Q 1 and Q 2 are each independently —O—C(O)—, —C(O)—O—, —O—C(S)—, —C(S)—O—, or —S—S—; and R 6 and R 7 are each independently C 1 -C 32 alkyl. A liquid composition comprising an ionized lipid of

12. The ionizable lipid is 【Chemistry 13】 、 【Chemistry 14】 、 【Chemistry 15】 、 【Chemistry 16】 、 【Chemistry 17】 、 [Chemistry 18] 、 【Chemistry 19】 , and 【Chemistry 20】 12. The liquid composition of claim 11, selected from the group consisting of:

13. 12. A method for making the liquid composition of claim 11, comprising mixing the freeze-dried RNA with the LNPs, wherein the LNPs are in a liquid solution and are added to the freeze-dried RNA.

14. The composition of claim 11 for treating a disease or disorder.

15. (i) Formula I: 【Chemistry 21】 [In the formula, R １ and R ２ are each independently １ -C ６ is alkyl; R ３ is C １ -C ５ is alkyl; Q １ , Q ２ , and Q ３ are each independently —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; L is C １ -C ３ is alkyl; R ４ and R ５ are each independently １ -C １０ is alkyl; R ６ and R ７ are each independently １ -C １０ Alkyl, C １ -C １０ alkenyl; A １ and A ２ are each independently a bond, —O—, —S—, —C(O)O—, —OC(O)—, —S—S—, —C(O)S—, —SC(O)—, —OC(S)—, or —C(S)O—; and R ８ and R ９ are each independently １ -C ３０ alkyl] ionized lipids; (ii) Formula II: 【Chemistry 22】 [In the formula, R 1 and R 2 are each independently C 1 -C 6 alkyl; R 3 is C 1 -C 5 alkyl; R 4 and R 5 are each independently a C 1 -C 18 alkyl group; Q 1 and Q 2 are each independently —O—C(O)—, —C(O)—O—, —O—C(S)—, —C(S)—O—, or —S—S—; and R 6 and R 7 are each independently C 18 -C 32 alkyl. or a pharmaceutically acceptable salt thereof; or (iii) 【Chemistry 23】 、 【Chemistry 24】 、 【Chemistry 25】 、 【Chemistry 26】 、 【Chemistry 27】 、 【Chemistry 28】 、 【Chemistry 29】 , and 【Transformation 30】 an ionizable lipid selected from the group consisting of Including, lipids.

16. 16. The lipid of claim 15, wherein the lipid is a lipid nanoparticle (LNP), the LNP further comprising a phospholipid, a structured lipid, and a PEGylated lipid.

17. The lipid of claim 16, wherein the lipid is a lipid nanoparticle (LNP), and the LNP comprises the phospholipid, the structured lipid, the PEGylated lipid, and the ionizable lipid in molar ratios ranging from 5% to 20% phospholipid, 18.5% to 58.5% structured lipid, 1% to 4% PEGylated lipid, and 30% to 70% ionizable lipid, wherein the sum of the molar ratios of the lipids is 100%.

18. The kit of claim 3, wherein the freeze-dried polynucleotide comprises a polyA tail of 30 to 100 nucleotides in length.

19. The kit of claim 3, wherein the lyophilized polynucleotide comprises a cap structure selected from the group consisting of m7G (cap 0), m7GpppNm- (cap 1), N6,2'-O-dimethyladenosine (m6AM), m7G(5')ppp(5')G (mCAP), and anti-reverse cap analog (ARCA), where Nm represents any 2'O-methylated nucleotide.

20. The kit of claim 3, wherein the freeze-dried polynucleotide comprises a nucleotide sequence encoding a varicella-zoster virus (VZV) antigen.

21. The freeze-dried polynucleotide, (i) a Cap1 cap, a VZV glycoprotein E (gE) antigen, a 3′UTR comprising approximately 270 nucleotides in length, and a poly-A tail comprising approximately 65 nucleotides in length; or (ii) a Cap1 cap, a SARS-CoV-2 spike protein (RBD), a 3'UTR comprising approximately 270 nucleotides in length, and a poly-A tail comprising approximately 65 nucleotides in length; The kit of claim 3, comprising:

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