Lyophilized mRNA-LNP vaccine product

Abstract

The application described herein provides a lyophilized RNA-LNP (e.g., mRNA-lipid nanoparticle), and methods of making or using the same, such as for vaccination using mRNA encoding an antigen vaccine (e.g., SARS-CoV-2).

Description

Background Technology

[0001] Messenger RNA (mRNA) is a single-stranded, anionic RNA molecule that, once it reaches the cytoplasm of a target cell, influences the expression of desired proteins (e.g., disease-specific antigens). To date, mRNA has shown great potential in vaccine and therapeutic applications, although efficient intracellular delivery of mRNA remains a major challenge.

[0002] Lipid nanoparticles (LNPs) have emerged as one of the most promising nonviral delivery vectors. LNPs encapsulate and protect fragile mRNA cargo from degradation, and promote its intracellular uptake and expression.

[0003] Currently, the only FDA-approved mRNA product is an mRNA vaccine for COVID-19 (e.g., mRNA-1273 and BNT162b2), in which anionic mRNA is encapsulated within a liquid nucleus (LNP) through electrostatic interactions with a cationic / ionizable lipid component, thereby producing an LNP-encapsulated mRNA (referred to herein as "mRNA-LNP") liquid dispersion as a vaccine product.

[0004] However, due to the inherent instability of mRNA, such LNP-encapsulated mRNA liquid vaccine products require cold chain management and ultra-low temperature storage, which greatly limits their global application.

[0005] Therefore, there is still a need for mRNA-based vaccine products that do not rely on such stringent storage and management conditions. Summary of the Invention

[0006] The present invention described herein provides lyophilized mRNA-LNP products that overcome the aforementioned challenges. Lyophilized LNP-encapsulated mRNA products are produced by formulating mRNA-LNPs into a lyophilized form using lyophilization protectants and other excipients in the established lyophilization method described herein. A method for producing such lyophilized LNP-encapsulated mRNA products is also provided.

[0007] In some embodiments, the lyophilized LNP-encapsulated mRNA product prepared according to the present invention comprises two components during application: a reconstitution solution (typically sterile water for injection) stored at, for example, room temperature; and lyophilized mRNA-LNP stored at 2°C to 8°C. In use, the reconstitution solution is added to the lyophilized mRNA-LNP, and then they are mixed to produce a homogeneous mRNA-LNP final product for administration.

[0008] Although the final vaccine product is manufactured differently from conventionally formulated LNP-encapsulated mRNA products (mRNA vaccines), the product developed using the method of this invention is as safe and effective as the widely used mRNA-LNP liquid product stored in frozen liquid.

[0009] More detailed embodiments of the invention are provided in the following numbered paragraphs.

[0010] 1. A lyophilized RNA-LNP (RNA-lipid nanoparticle, e.g., mRNA-LNP), wherein the RNA-lipid nanoparticle comprises RNA (e.g., mRNA) encapsulated in a lipid nanoparticle (LNP), and wherein the LNP comprises an ionizable lipid having the structure of formula (I):

[0011] Formula (I), in, R1 and R2 are independently C1-3 alkyl groups; R3 is C 1-4 alkyl; R4 and R5 are independently C1- 10 alkyl; Q1, Q2 and Q3 are each independently -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O- or -SS-; L stands for -(CH2) 0-2 -CH<or (For example, R4 and R5 are connected to L via -O-). R6 and R7 are independently C1- 10 Alkyl or C1- 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-, or -SS-, and R8 and R9 are independently C1- 10 Alkyl or C1- 10 alkenyl; Optionally, R6 contains at least one (e.g., 1) double bond, R7 contains at least one (e.g., 1) double bond, R8 contains at least one (e.g., 1 or 2) double bond, and / or R9 contains at least one (e.g., 1 or 2) double bond.

[0012] 2. The lyophilized RNA-LNP according to paragraph 1, wherein the LNP further comprises: (1) Sterol lipids (e.g., cholesterol); (2) Phospholipids (e.g., DSPC), and (3) Polyethylene glycol-modified lipids (e.g., DMG-PEG2000).

[0013] 3. The freeze-dried RNA-LNP as described in paragraph 1 or 2, wherein the moisture content of the freeze-dried RNA-LNP is < about 5% (e.g., < about 3%).

[0014] 4. The lyophilized RNA-LNP according to any one of paragraphs 1 to 3, wherein the lyophilized RNA-LNP is stable under storage conditions of 2°C to 8°C (e.g., after 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks, the loss of biological activity (such as transfection efficiency) of the RNA-LNP does not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%).

[0015] 5. The lyophilized RNA-LNP according to any one of paragraphs 1 to 4, wherein when the lyophilized RNA-LNP is reconstituted in a reconstitution solution (e.g., sterile water for injection or WFI), the lyophilized RNA-LNP is substantially free of visible particles.

[0016] 6. The lyophilized RNA-LNP as described in paragraph 5, wherein the RNA concentration of the reconstituted lyophilized RNA-LNP is at least about 60 μg / mL (RNA equivalent).

[0017] 7. Lyophilized RNA-LNP according to any one of paragraphs 1 to 6, wherein, after reconstitution, substantially all (e.g., >80%, >85%, >90%, >90%, or >95% of all particles) of particles are <250 nm in size (e.g., <200 nm, <190 nm, <180 nm, <170 nm, <160 nm, or <150 nm).

[0018] 8. The lyophilized RNA-LNP according to any one of paragraphs 1 to 7, wherein the combined molar ratio of said ionizable lipid and said cholesterol is about 85% to 90%, for example about 88%.

[0019] 9. The lyophilized RNA-LNP according to any one of paragraphs 1 to 8, wherein the molar ratio of said ionizable lipid is about 25 mol% to 70 mol%, about 35 mol% to 60 mol%, about 35 mol% to 55 mol%, or about 40 mol% to 55 mol%.

[0020] 10. The lyophilized RNA-LNP according to any one of paragraphs 1 to 9, wherein the molar ratio of the ionizable lipid, the cholesterol, the phospholipid and the polyethylene glycol-modified lipid is about 40:48:10:2.0.

[0021] 11. A lyophilized RNA-LNP according to any one of paragraphs 1 to 10, wherein R1 and R2 are both -Me.

[0022] 12. The lyophilized RNA-LNP according to paragraph 11, wherein Q1 is -OC(O)- or -C(O)-O-, and is linked to L by -O- or -C(O)-.

[0023] 13. The lyophilized RNA-LNP as described in paragraph 12, wherein L is -CH2-CH<; R4 and R5 are both -(CH2)7-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 is C9-alkenyl; R7 is C 8-9 Alladienyl; A1, A2, and R8 are all bonds; and R9 is C. 7-8 Alkenyl group.

[0024] 14. The lyophilized RNA-LNP according to paragraph 13, wherein the ionizable lipid has the following structure:

[0025] (Lipid No. 10).

[0026] 15. The lyophilized RNA-LNP according to paragraph 12, wherein L is -CH<; R4 and R5 are both -(CH2)8-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 and R7 are both C6-alkenyl; A1 and A2 are both -O-; and R8 and R9 are both -(CH2)6-.

[0027] 16. The lyophilized RNA-LNP according to paragraph 15, wherein the ionizable lipid has the following structure:

[0028] (Lipid No. 11).

[0029] 17. A lyophilized RNA-LNP according to any one of paragraphs 1 to 16, wherein the RNA: (a) is a self-replicating RNA (e.g., mRNA) or a non-self-replicating RNA (e.g., mRNA), and / or (b) Encoding immunogenic antigens (e.g., one or more immunogenic antigens derived from the same or different proteins).

[0030] 18. The lyophilized RNA-LNP as described in paragraph 17, wherein the immunogenic antigen includes viral proteins or cancer antigens.

[0031] 19. The freeze-dried RNA-LNP according to paragraph 18, wherein the viral protein is the spike protein (e.g., S-2P protein) of SARS-CoV-2 virus (e.g., the Omecron variant of said SARS-CoV-2 virus) or an antigenic fragment thereof.

[0032] 20. A method for producing lyophilized RNA-LNP according to any one of paragraphs 1 to 19, the method comprising lyophilizing a liquid dispersant comprising the RNA-lipid nanoparticles.

[0033] 21. The method according to paragraph 20, wherein the liquid dispersant is formulated by mixing an organic phase (e.g., an ethanol phase) with an aqueous phase using a microfluidic device, wherein: (1) The organic phase (e.g., the ethanol phase) comprises: ionizable lipids, cholesterol, phospholipids (e.g., 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC)) and polyethylene glycol-modified lipids (e.g., 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000)); and (2) The aqueous phase contains RNA dissolved in an aqueous buffer (such as 50 mM citrate buffer (pH 6.0)).

[0034] 22. The method according to paragraph 20 or 21, further comprising purifying the liquid dispersant in an aqueous buffer containing a protectant (e.g., sucrose) prior to lyophilization, and adjusting the pH to a predetermined level (e.g., pH 7.2) if necessary.

[0035] 23. The method according to paragraph 22, the method further comprising adjusting the concentration of the liquid dispersant prior to freeze-drying and / or storing the liquid dispersant at 2°C to 8°C.

[0036] 24. The method according to any one of paragraphs 20 to 23, wherein the liquid dispersant is freeze-dried by a freeze-drying process comprising pre-freezing, primary drying and secondary drying.

[0037] 25. A lyophilized RNA-LNP produced by the method according to any one of paragraphs 20 to 24.

[0038] 26. A pharmaceutical composition comprising lyophilized RNA-LNP (e.g., mRNA-LNP) according to any one of paragraphs 1 to 19 and 25 and a pharmaceutically acceptable excipient.

[0039] 27. The pharmaceutical composition according to paragraph 26, wherein the pharmaceutical composition is formulated for use in vaccination (e.g., human vaccination).

[0040] 28. A kit comprising (a) lyophilized RNA-LNP according to any one of paragraphs 1 to 19 and 25, or a pharmaceutical composition according to paragraph 27 or 28; and (b) an aqueous reconstitution solution (e.g., sterile water for injection).

[0041] 29. The kit according to paragraph 28, wherein the lyophilized RNA-LNP package is for storage at 2°C to 8°C, and wherein the aqueous reconstitution solution package is for storage at room temperature (RT or 20°C to 25°C).

[0042] 30. A method for reconstituted lyophilized RNA-LNP according to any one of paragraphs 1 to 19 and 25, the method comprising adding an aqueous liquid to the lyophilized RNA-LNP.

[0043] 31. The method according to paragraph 30, wherein the aqueous liquid is sterile water for injection (WFI).

[0044] 32. A method of immunizing a mammal, the method comprising administering to the mammal lyophilized mRNA-LNP (RNA-lipid nanoparticles) reconstituted in sterile water for injection (WFI) according to any one of paragraphs 1 to 19 and 25, wherein the RNA encodes an antigen vaccine.

[0045] 33. According to the method described in paragraph 32, the mammal is a human (e.g., a human child aged 6 months to about 4 to 5 years; a human child aged 5 or 6 to 11 years; a human child aged 12 to 17 years; or a human adult aged 18 years and older).

[0046] 34. The method according to paragraph 32 or 33, wherein the RNA is mRNA encoding a spike protein (e.g., S-2P protein) or an antigenic fragment of the SARS-CoV-2 virus (e.g., an Omeprone variant of the SARS-CoV-2 virus).

[0047] It should be understood that any of the embodiments described herein, including those described only in the embodiments or claims, may be combined with any one or more other embodiments of this disclosure, unless such combination is inappropriate or expressly denied. Attached Figure Description

[0048] Figure 1The expression levels of SARS-CoV-2 spike protein in BHK-21 cells (left panel) and Vero E6 cells (right panel) are shown at 24, 48, and 72 hours after transfection with reconstituted lyophilized mRNA-LNP product or mRNA-LNP intermediate control (produced without lyophilization).

[0049] Figure 2 The immunization protocol and blood collection schedule in laboratory animals (mice or cynomolgus monkeys) are shown for testing antibody titers after immunization with a vaccine test item that is either a control or a reconstituted lyophilized mRNA-LNP product.

[0050] Figure 3 The results of immunization of balb / c mice in the vaccine test program described in Example 6 are shown, as measured by the geometric mean titer (GMT) of the anti-SARS-CoV-2 spike protein antibody.

[0051] Figure 4 The results of immunization of cynomolgus monkeys in the vaccine test program described in Example 6 are shown, as measured by the geometric mean titer (GMT) of the anti-SARS-CoV-2 spike protein antibody.

[0052] Figure 5 The structures of lipids 11, 10, 2, 4 and Dlin-MC3-DMA are shown.

[0053] Figure 6 The in vitro transfection efficiencies of various S2P mRNA-LNP products containing different ionizable lipids (formulation IDs 1 to 5 for lanes 1 to 5) are shown, as measured by Western blot (WB) detection of RBD expression. Detailed Implementation

[0054] 1. Overview The present invention described herein provides a method for producing lyophilized mRNA-LNPs that can be stored stably for an extended period of time at approximately 2°C to 8°C. The invention also provides lyophilized mRNA-LNPs produced in such a way that, upon reconstitution, the resulting final product exhibits substantially the same efficacy level in animals as a vaccine, compared to mRNA-LNP liquid dispersants manufactured using standard methods (i.e., methods without lyophilization).

[0055] Aside from the largely unchanged vaccine efficacy, the unique manufacturing method described in this article allows mRNA vaccines to be stored under widely accepted conditions for global use, providing great flexibility to vaccine providers, especially those in countries or regions that have difficulty accessing high-end cryopreservation facilities.

[0056] Importantly, the invention described herein is not limited to SARS-CoV-2 mRNA vaccines, but has broad and general applicability to a variety of mRNA active pharmaceutical ingredients. Therefore, the invention described herein can be used to produce a variety of mRNA (e.g., vaccine) products.

[0057] Therefore, one aspect of the present invention described herein provides a lyophilized RNA-LNP (RNA-lipid nanoparticle, e.g., mRNA-LNP), wherein the RNA-lipid nanoparticle comprises RNA (e.g., mRNA) encapsulated in a lipid nanoparticle (LNP), and wherein the LNP comprises an ionizable lipid having the structure of formula (I):

[0058] Formula (I), in, R1 and R2 are independently C1-3 alkyl groups; R3 is C 1-4 alkyl; R4 and R5 are independently C1- 10 alkyl; Q1, Q2 and Q3 are each independently -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O- or -SS-; L stands for -(CH2) 0-2 -CH<or (For example, R4 and R5 are connected to L via -O-). R6 and R7 are independently C1- 10 Alkyl or C1- 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-, or -SS-, and R8 and R9 are independently C1- 10 Alkyl or C1- 10 alkenyl; Optionally, R6 contains at least one (e.g., 1) double bond, R7 contains at least one (e.g., 1) double bond, R8 contains at least one (e.g., 1 or 2) double bond, and / or R9 contains at least one (e.g., 1 or 2) double bond.

[0059] In some embodiments, the LNP also includes: (1) sterol lipids (e.g., cholesterol); (2) phospholipids (e.g., DSPC); and (3) PEGylated lipids (e.g., DMG-PEG2000).

[0060] In some implementations, L in equation (I) above is -(CH2). 0-2 -CH<, such as -(CH2)-CH< or -CH<, where R4 and R5 are connected to L via –CH<.

[0061] In some implementations, R6 and R7 are the same. In some implementations, R6 and R7 are different. In some implementations, R6 and R7 are each (the same or different) C1- 10 Alkenyl groups, such as (same or different) straight-chain C1- 10 Alkenyl group. In some embodiments, R6 and R7 are each (identical or different) C1- groups having a double bond. 10 Alkenyl. In some embodiments, R6 and R7 are each (same or different) a C6 alkenyl having a double bond. In some embodiments, R6 and R7 are each a straight-chain C6 alkenyl (hexene) (2-hexene) having a double bond between the second and third carbon atoms of Q2 or Q3.

[0062] In some implementations, R8 and R9 are the same. In some implementations, R8 and R9 are different. In some implementations, R8 and R9 are each (the same or different) C1- 10 Alkyl groups, such as (same or different) straight-chain C1- 10 Alkyl group. In some embodiments, R8 and R9 are each C10. 6-8 Alkyl group. In some embodiments, R8 and R9 are each a straight-chain C5 alkyl group, a straight-chain C6 alkyl group, or a straight-chain C7 alkyl group.

[0063] In some implementations, R6 and R7 are the same, and R8 and R9 are the same. In some implementations, R6 and R7 are each the same linear C1- 10 Alkenyl groups, and R8 and R9 are each the same straight-chain C1- 10 Alkyl group. In some embodiments, R6 and R7 are each having a double bond (e.g., in Q). 2 Or Q 3 The same straight-chain C6 alkenyl group (between the second and third carbon atoms), and R8 and R9 are each C6, C7 or C8 alkyl groups.

[0064] In some embodiments, R6 and R7 are different. In some embodiments, R6-A1-R8 together and / or R7-A2-R9 together each form a straight-chain or branched alkylene group. In some embodiments, R6-A1-R8 together form a straight-chain alkylene group, and R7-A2-R9 together each form a branched alkylene group. In some embodiments, R6-A1-R8 together form a straight-chain alkylene group having one double bond, and R7-A2-R9 together form a branched alkylene group having two symmetrical or asymmetrical straight-chain branches, each branch having one double bond. In some embodiments, R6-A1-R8 together form a straight-chain C group having one double bond (such as the double bond between the second and third carbon atoms of Q2). 8-10 Alkylenes (such as C9 alkylenes), and R7-A2-R9 together form a branched alkylene with two identical and symmetrical straight-chain branches, wherein the branched carbon is adjacent to Q3, and wherein each branch has a double bond (e.g., between the 4th and 5th carbon atoms from the branched carbon).

[0065] In some implementations, L in equation (I) above is (For example, R4 and R5 are connected to L via -O-). In some embodiments, R4 and R5 are the same. In some embodiments, R4 and R5 are different. In some embodiments, R4 and R5 are each the same straight-chain C1- 10 Alkyl groups, such as C6 alkyl, straight-chain C7 alkyl, or straight-chain C8 alkyl. In some embodiments, R6 and R7 are the same. In some embodiments, R6 and R7 are different. In some embodiments, R6 and R7 are each the same straight-chain C1- 10 Alkyl groups, such as C8 alkyl, straight-chain C9 alkyl, or straight-chain C 10 Alkyl group. In some embodiments, A1 and A2 are each a bond.

[0066] In some implementations, L in equation (I) above is (For example, R4 and R5 are connected to L via -O-). In some embodiments, R4 and R5 are the same. In some embodiments, R4 and R5 are different. In some embodiments, R4 and R5 are each the same straight-chain C1- 10 Alkyl groups, such as C6 alkyl, straight-chain C7 alkyl, or straight-chain C8 alkyl. In some embodiments, R6 and R7 are different. In some embodiments, R6-Al-R8 together form a straight-chain C6 alkyl group. 8-10 Alkyl groups (such as C9 alkyl groups), and R7-A2-R9 together form a branched alkyl group having two identical and symmetrical straight-chain branches, optionally with the branched carbon adjacent to Q3, and optionally, each branch is a straight-chain C. 7-9 Alkyl groups (such as C8 alkyl groups).

[0067] In some embodiments, the ionizable lipid of formula (I) contains no more than three (3) double bonds. In some embodiments, the ionizable lipid of formula (I) contains a total of two double bonds. In some embodiments, the ionizable lipid of formula (I) contains a total of three double bonds. In some embodiments, each of R6 and R7 has no more than one double bond. In some embodiments, R6-A1-R8 together have no more than two double bonds. In some embodiments, R7-A2-R9 together have no more than two double bonds.

[0068] In some implementations, both R1 and R2 are -Me.

[0069] In some implementations, Q1 is -OC(O)- or -C(O)-O-, and is connected to L via -O- or -C(O)-.

[0070] In some embodiments, L is -CH<; R4 and R5 are both -(CH2)8-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 and R7 are both C6-alkenyl; A1 and A2 are both -O-; and R8 and R9 are both -(CH2)6-.

[0071] In some embodiments, the ionizable lipid has the following structure:

[0072] (Lipid No. 11).

[0073] In some embodiments, L is -CH2-CH<; R4 and R5 are both -(CH2)7-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 is C9-alkenyl; R7 is C 8-9 Alladienyl; A1, A2, and R8 are all bonds; and R9 is C. 7-8 Alkenyl group.

[0074] In some embodiments, the ionizable lipid has the following structure:

[0075] (Lipid No. 10).

[0076] In some implementations, the ionizable lipid is lipid number 2.

[0077] In some implementations, the ionizable lipid is lipid number 4.

[0078] In some implementations, C 1-10The alkyl group is methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or their branched variants (such as isopropyl, 3-pentyl, secondary pentyl, etc.).

[0079] In some implementations, C 1-10 The alkenyl group is ethylene, propylene, 1-butene, 2-butene, isobutene, 1-pentene, 2-pentene, 2-methyl-1-butene, 3-methyl-1-butene, 2-methyl-2-butene, 1-hexene, 2-hexene, 3-hexene, 2-methyl-1-pentene, 3-methyl-1-pentene, 4-methyl-1-pentene, 2-methyl-2-pentene, 3-methyl-2-pentene, 4-methyl-2-pentene, 2,3-dimethyl-1-butene, 3,3-dimethyl-1-butene, 2,3-dimethyl-2-butene, 2-ethyl-1-butene, etc. In some embodiments, the alkenyl group contains 1, 2, 3, 4, or 5 double bonds. In some embodiments, each alkenyl group contains one double bond.

[0080] In some embodiments, the phospholipids include phosphatidylcholine, such as distearylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and / or phosphate ethanolamine, such as 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine (DSPE) or 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0081] Glycerolipids are a class of phospholipids containing a hydrophilic head group and two hydrophobic fatty acyl tails linked to the glycerol backbone. The hydrophilic head determines the charge of the LNP particle, which can theoretically be neutral, anionic (negative), or cationic (positive). In some embodiments, the phospholipids in the LNP are anionic lipids, such as phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), or phosphatidic acid (PA).

[0082] In some embodiments, the phospholipids include: (DSPC) (DSPE) (DOPE) or (DOPC).

[0083] In some implementations, steroids comprise sterol lipids (such as cholesterol), and are essentially composed of or consist of sterols.

[0084] While not wishing to be bound by any particular theory, sterol lipids (such as cholesterol) can fill stacking defects in lipid membranes and provide structural integrity for tested LNPs. They can also facilitate membrane fusion between LNPs and target cells.

[0085] In some embodiments, sterol lipids include sterols and / or open-ring sterols, such as animal sterols, phytosterols, and fungal sterols, as well as combinations and mixtures thereof. In some embodiments, sterol lipids include cholesterol and / or phytosterols, and / or particularly at least one open-ring sterol, preferably at least calciferol (vitamin D); particularly wherein the sterols and / or open-ring sterols (especially sterols) have a Griffin HLB value of up to 1.5, and / or particularly wherein the sterols and / or open-ring sterols (especially sterols) have a log P value of at least 5.

[0086] In some implementations, sterol lipids are cholesterol, cholesterol sulfate, or their derivatives, such as 7α-hydroxycholesterol (which improves the delivery of oligonucleotide cargoes).

[0087] cholesterol Cholesterol sulfate Furthermore, while not wishing to be bound by any particular theory, the PEGylated lipids in the LNPs described herein prevent serum protein adsorption and inhibit uptake by the mononuclear phagocyte system (MPS), a major obstacle to LNP delivery. In some embodiments, the PEGylated lipids also contain terminal functional groups, such as amines or maleimides, which can be used to conjugate other molecules that improve cell targeting and uptake.

[0088] In some embodiments, the PEGylated lipids / stealth lipids comprise PEGylated lipids having PEG (-O-CH2-CH2-) repeats, such as DMG-PEG2000 ( ), which is basically composed of or made up of.

[0089] In some embodiments, the PEGylated lipids / stealth lipids comprise, are substantially composed of, or are composed of C-DMG-PEG(2000) / PEG(2000)-C-DMG or ALC-0159.

[0090] The ionizable lipids described herein that can be used in this invention can be prepared based on, for example, WO2022166213A1 and WO2023104114A2 (incorporated herein by reference).

[0091] In some embodiments, the moisture content of the freeze-dried RNA-LNP is < about 5% (e.g., < about 3%).

[0092] In some embodiments, the lyophilized RNA-LNPs described herein are stable under storage conditions from 2°C to 8°C. In some embodiments, the loss of biological activity (such as transfection efficiency) of the RNA-LNPs does not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% after 2, 3, 4, 5, or 6 weeks.

[0093] In some embodiments, when the lyophilized RNA-LNP is reconstituted in a reconstitution solution (e.g., sterile water for injection or WFI), the lyophilized RNA-LNP described herein is substantially free of visible particles.

[0094] In some implementations, the reconstituted lyophilized RNA-LNP has an RNA concentration of at least about 60 μg / mL (RNA equivalent).

[0095] In some embodiments, after resolution, substantially all (e.g., >80%, 85%, 90%, 90%, 95% of all particles) of particles have a size <250 nm (e.g., <200 nm, <190 nm, <180 nm, <170 nm, <160 nm, or <150 nm).

[0096] In some embodiments, the lyophilized RNA-LNPs described herein have a polydispersity index (PDI) between about 0.10 and 0.25 or between about 0.15 and 0.20 before lyophilization or after lyophilization and reconstitution.

[0097] In some implementations, the combined molar ratio of ionizable lipids and cholesterol is about 85% to 90%, for example, about 88%.

[0098] In some embodiments, the molar ratio of ionizable lipids is about 25 mol% to 70 mol%, about 35 mol% to 60 mol%, about 35 mol% to 55 mol%, or about 40 mol% to 55 mol%.

[0099] In some embodiments, the lyophilized RNA-LNP described herein comprises: (a) about 25 mol% to 70 mol%, about 35 mol% to 60 mol%, about 35 mol% to 55 mol%, or about 40 mol% to 55 mol% of ionizable lipids in molar percentage; (b) about 5 mol% to 20 mol% (such as about 5 mol% to 15 mol% or about 10 mol%) of phospholipid composition in molar percentage; (c) about 35 mol% to 60 mol% (such as about 45 mol% to 50 mol% or about 48 mol%) of steroids (such as cholesterol) in molar percentage; and (d) about 0 mol% to 5 mol% (such as about 1 mol% to 3 mol% or about 2 mol%) of stealth lipids / polyethylene glycol-modified lipids (such as DMG-PEG2000) in molar percentage.

[0100] In some implementations, the molar ratio of ionizable lipids, cholesterol, phospholipids and polyethylene glycol-modified lipids is approximately (±0.5) 40:48:10:2.0.

[0101] In some implementations, the N / P molar ratio of the lyophilized RNA-LNP is 6, 7, or 8.

[0102] As used herein, “N / P molar ratio” refers to the ratio of the molar amount of charged nitrogen (N) in an ionizable lipid composition to the molar amount of phosphorus (P) in an LNP formulation of RNA polynucleotides.

[0103] In some embodiments, the RNA in the lyophilized RNA-LNP described herein is: (a) a self-replicating RNA (e.g., mRNA), a non-self-replicating RNA (e.g., mRNA), or a circular RNA, and / or (b) encodes an immunogenic antigen (e.g., one or more immunogenic antigens derived from the same or different proteins).

[0104] In some implementations, RNA is siRNA, miRNA, pri-miRNA, messenger RNA (mRNA), clustered regularly spaced short palindromic repeats (CRISPR) related nucleic acid, single-stranded guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), transfer RNA (tRNA), antisense oligonucleotide (ASO), guide RNA, single-stranded RNA (ssRNA), or double-stranded RNA (dsRNA).

[0105] In some implementations, the immunogenic antigen includes viral proteins or cancer antigens.

[0106] In some implementations, the viral protein is the spike protein (e.g., S-2P protein) of the SARS-CoV-2 virus (e.g., the Omecron variant of the SARS-CoV-2 virus) or an antigenic fragment thereof.

[0107] Another aspect of the invention provides a method for producing the lyophilized RNA-LNPs described herein, the method comprising lyophilizing a liquid dispersant comprising the RNA-lipid nanoparticles described herein.

[0108] In some embodiments, a liquid dispersion is formulated by mixing an organic phase (e.g., an ethanol phase) with an aqueous phase using a microfluidic device, wherein: (1) the organic phase (e.g., the ethanol phase) comprises: ionizable lipids, cholesterol, phospholipids (e.g., 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC)) and polyethylene glycol-modified lipids (e.g., 1,2-dimyristoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000)); and (2) the aqueous phase comprises RNA dissolved in an aqueous buffer (such as 50 mM citrate buffer (pH 6.0)).

[0109] In some embodiments, the method further includes purifying the liquid dispersant in an aqueous buffer containing a protectant (e.g., sucrose) prior to lyophilization, and adjusting the pH to a predetermined level (e.g., pH 7.2) if necessary.

[0110] In some embodiments, the method further includes adjusting the concentration of the liquid dispersant prior to freeze-drying and / or storing the liquid dispersant at 2°C to 8°C.

[0111] In some embodiments, the liquid dispersant is freeze-dried via a process that includes pre-freezing, primary drying, and secondary drying.

[0112] Another aspect of the invention provides lyophilized RNA-LNPs produced by the methods described herein.

[0113] Another aspect of the invention provides a pharmaceutical composition comprising the lyophilized RNA-LNP (e.g., mRNA-LNP) described herein and a pharmaceutically acceptable excipient or carrier.

[0114] Pharmaceutical compositions / formulations can be prepared by any method known or subsequently developed in the field of pharmaceutical science. Typically, such preparation methods involve the following steps: associating an active ingredient (e.g., the LNP formulation described herein, prior to lyophilization) with one or more excipients and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into desired single-dose or multi-dose units.

[0115] In some embodiments, the pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, as used herein, including any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired particular dosage form. Remington's *The Science and Practice of Pharmacy*, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient is incompatible with the substance or its derivatives (e.g., by producing any undesirable biological effects or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition), its use is considered to be within the scope of this invention.

[0116] In some embodiments, the pharmaceutically acceptable excipient has a purity of at least 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient conforms to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0117] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients may optionally be included in pharmaceutical formulations containing the immunogenic nanoparticles described herein. Excipients (such as cocoa butter and suppository waxes), colorants, coating agents, sweeteners, flavoring agents, and aromatizers may be present in the composition at the discretion of the formulator.

[0118] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and combinations thereof.

[0119] Exemplary granulating agents and / or dispersants include, but are not limited to, potato starch, corn starch, cassava starch, sodium hydroxymethyl starch, clay, alginate, guar gum, citrus pomace, agar, bentonite, cellulose and wood products, natural sponges, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crosspovidone), sodium carboxymethyl starch (sodium hydroxymethyl starch), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (cross-linked carboxymethyl cellulose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and combinations thereof.

[0120] Exemplary surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth gum, chondroitin, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, waxes, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, glyceryl triacetate monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxylated polymethylene, polyacrylic acid, acrylic polymers and carboxylated vinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethyl cellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose), and sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol monolaurate [TWEEN20®]). Polyoxyethylene sorbitol [TWEEN60®], polyoxyethylene sorbitol monooleate [TWEEN80®], sorbitol monopalmitate [SPAN40®], sorbitol monostearate [SPAN®60], sorbitol tristearate [SPAN®65], glyceryl monooleate, sorbitol monooleate [SPAN®80], polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®4]). 5], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ®30]), polyvinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, poloxamer 188, cetrimonium bromide, cetrimonium chloride, benzalkonium chloride, sodium docusate, etc., and / or combinations thereof.

[0121] Exemplary disintegrants include, but are not limited to, agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate.

[0122] Exemplary adhesives include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., gum arabic, sodium alginate, extracts of Irish moss, panwa gum, Indian gum, isapour, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum), and larch arabinogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethyl methacrylate; waxes; water; alcohols, etc., and combinations thereof.

[0123] Exemplary preservatives may include antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbate palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citrate monohydrate, disodium edetate, dipotassium edetate, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzyl alcohol, bromonitrile glycol, cetrimonium bromide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethanol, glycerin, hexoterein, imidazoline, phenol, phenoxyethanol, phenethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid. Exemplary alcoholic preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, parabens, and phenethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deferoxamine mesylate, cetrimonium bromide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, Germall 115, Germaben II, Neolone™, Kathon™, and EUXYL®. In some embodiments, the preservative is an antioxidant. In other embodiments, the preservative is a chelating agent.

[0124] Exemplary buffers include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium acetopropionate, valeric acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dibasic potassium phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, magnesium hydroxide, aluminum hydroxide, alginate, pyrogen-free raw water, isotonic saline, Ringer's solution, ethanol, etc., and combinations thereof.

[0125] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0126] Exemplary oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, canola oil, caraway oil, carnation oil, Brazil palm oil, castor oil, cinnamon oil, cocoa butter, coconut oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, flaxseed oil, geraniol oil, gourd oil, grapeseed oil, hazelnut oil, hyssop oil, isopropyl myristate, jojoba oil, coukow oil, and mixed lavender oil. Lavender oil, lemon oil, litsea cubeba oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange salmon oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, tea plum oil, peppermint oil, sea buckthorn oil, sesame oil, shea butter, silicone oil, soybean oil, sunflower seed oil, tea tree oil, milk thistle oil, ailanthus oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, caprylic triglyceride, cyclomethyl silicone, diethyl sebacate, dimethyl silicone 360, isopropyl myristate, mineral oil, octyl dodecyl alcohol, oleyl alcohol, silicone oil, and combinations thereof.

[0127] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters and mixtures thereof of sorbitol. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and aromatizers. In some embodiments for parenteral application, the therapeutic agent of the present invention is mixed with solubilizers such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and combinations thereof.

[0128] Injectable formulations (e.g., sterile injectable aqueous or oily suspensions) can be formulated using suitable dispersants or wetting agents and suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, or emulsions in non-toxic, parenteral-acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable solvents and media that can be used are water, Ringer's solution, USP, and isotonic sodium chloride solution. Furthermore, sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid are used in the preparation of injectable formulations.

[0129] Injectable formulations can be sterile, for example, filtered through a bacterial trap filter, or incorporated into a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0130] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and silicate; b) binders such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; c) wetting agents such as glycerin; d) disintegrants such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; e) solution blocking agents such as paraffin; f) absorption enhancers such as quaternary ammonium compounds; g) humectants such as cetyl alcohol and glyceryl monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may contain a buffer.

[0131] Similar types of solid compositions can be used as fillers in soft-filled and hard-filled gelatin capsules that use excipients such as lactose or toffee and high molecular weight polyethylene glycol, etc. Solid dosage forms of tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shells (such as enteric coatings and other coatings well known in the field of pharmaceutical formulation). They may optionally contain opaque agents and can be a composition that optionally releases one or more active ingredients in a delayed manner only or preferably in a portion of the intestine. Examples of encapsulation compositions that can be used include polymeric substances and waxes. Similar types of solid compositions can be used as fillers in soft-filled and hard-filled gelatin capsules that use excipients such as lactose or toffee and high molecular weight polyethylene glycol, etc.

[0132] In some embodiments, the therapeutic agent encapsulated by tLCNP may be in a microencapsulated form together with one or more excipients as described above. Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules may be prepared using coatings and shells (such as enteric coatings, controlled-release coatings, and other coatings well known in the field of pharmaceutical formulation). In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent (such as sucrose, lactose, or starch). As is common practice, such dosage forms may contain additional substances besides inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pellets, the dosage form may contain a buffer. They may optionally contain an opaque agent and may be a composition that optionally releases the therapeutic agent in a delayed manner only or preferably in a portion of the intestine. Examples of encapsulation compositions that may be used include polymers and waxes.

[0133] In some embodiments, dosage forms for topical and / or transdermal application of therapeutic agents may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and / or patches. Typically, the active ingredient is mixed under aseptic conditions with pharmaceutically acceptable excipients and / or any desired preservatives and / or buffers (if necessary). Transdermal patches are also considered. Such patches generally offer the additional advantage of controlled delivery of the active ingredient to the body. For example, such dosage forms can be prepared by dissolving and / or dispersing the active ingredient in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlled membrane and / or by dispersing the active ingredient in a polymer matrix and / or gel.

[0134] Suitable devices for delivering the intradermal pharmaceutical compositions described herein include soluble microneedle array patches, such as those described in U.S. Patent Publications: US 2019 / 0358441 A1, US 2019 / 0240469 A1, US 2019 / 0151638 A1, US 2019 / 0151638 A1, US 2018 / 0001070 A1, US 2017 / 0209553 A1, US 2016 / 0263362 A1, US 2017 / 0196966 A1, US 2016 / 0213908 A1, US 2016 / 0015952 A1, US 2015 / 0112250 A1, US 2012 / 0150023, etc. Short-needle devices, such as those described in U.S. Patent Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662, are also considered. Intradermal compositions can be administered via devices that limit the effective penetration length of the needle into the skin, such as those described in PCT Publication WO 99 / 34850 and their functional equivalents. Jet injection devices that deliver vaccines to the dermis via a liquid jet injector and / or via a needle that pierces the stratum corneum and generates a jet reaching the dermis are suitable. Jet injection devices are described, for example, in U.S. Patent Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT Publications WO 97 / 037705 and WO 97 / 013537, etc. A ballistic powder / particle delivery device that uses compressed gas to accelerate the delivery of powdered vaccines through the outer layer of skin to the dermis is suitable. Alternatively or otherwise, a conventional syringe can be used for intradermal administration using the classic Mantoux method.

[0135] In some embodiments, formulations suitable for topical application include, but are not limited to, liquid and / or semi-liquid formulations (such as liniments, lotions), oil-in-water and / or water-in-oil emulsions (such as creams, ointments, and / or pastes), and / or solutions and / or suspensions. In some embodiments, a topically applicable formulation may, for example, contain about 1% to about 10% (w / w) of a therapeutic agent (the immunogenic nanoparticles described herein), although the concentration of the active ingredient may be up to the solubility limit of the active ingredient in a solvent. In some embodiments, formulations for topical application may also contain one or more additional ingredients described herein.

[0136] In some embodiments, pharmaceutical compositions comprising the tLCNP formulation described herein can be prepared, packaged, and / or marketed as formulations suitable for pulmonary administration via the buccal cavity. Such formulations may, for example, comprise particles containing or containing a therapeutic agent encapsulated with the tLCNP described herein. Such compositions are conveniently in dry powder form for administration using a device comprising a dry powder reservoir and / or a self-propelled solvent / powder dispensing container, the propellant flow of which can be directed to the dry powder reservoir to disperse the powder. This self-propelled solvent / powder dispensing container may include a device comprising dissolving and / or suspending the active ingredient in a low-boiling-point propellant within a sealed container.

[0137] Low-boiling-point propellants typically include liquid propellants with a boiling point below 65℉ at atmospheric pressure. In some embodiments, the propellant may constitute 50% to 99.9% (w / w) of the composition, and the therapeutic agent may constitute 0.1% to 20% (w / w) of the composition. The propellant may also contain additional components, such as liquid nonionic and / or solid anionic surfactants and / or solid diluents (these additional components may have a particle size on the same order of magnitude as the particles containing the active ingredient).

[0138] In some embodiments, pharmaceutical compositions comprising the therapeutic agents described herein, formulated for pulmonary delivery, may provide nanoparticles in the form of droplets as solutions and / or suspensions. In some embodiments, such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions (optionally aseptically) containing the active ingredient, and may be conveniently administered using any spray and / or nebulizer. In some embodiments, such formulations may also contain one or more additional ingredients, including but not limited to flavoring agents (such as sodium saccharin), volatile oils, buffers, surfactants, and / or preservatives (such as methylparaben). In some embodiments, the average diameter of the droplets delivered via this route of administration may be in the range of about 0.1 pm to about 200 pm.

[0139] In some embodiments, the formulations described herein for lung delivery can be used for intranasal delivery of the pharmaceutical compositions of the present invention. Another exemplary formulation suitable for intranasal administration is a coarse powder containing an active ingredient and having an average particle size of about 0.2 μm to about 500 pm. This formulation can be administered by nasal inhalation, i.e., rapid inhalation through the nasal passage from a powder container near the nostril.

[0140] In some embodiments, formulations suitable for intranasal administration may, for example, comprise as little as 0.1% (w / w) and as much as 100% (w / w) of the tLCNP formulation described herein, and may include one or more additional ingredients described herein. In some embodiments, pharmaceutical compositions of the tLCNP formulation described herein may be prepared, packaged, and / or marketed as formulations suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges prepared using conventional methods, and may, for example, comprise 0.1% to 20% (w / w) of the tLCNP formulation described herein, with the balance comprising an orally soluble and / or biodegradable composition and optionally one or more additional ingredients described herein. Alternatively, in some embodiments, formulations suitable for buccal administration may comprise powders containing immunogenic nanoparticles and / or aerosolized and / or nebulized solutions and / or suspensions. In some embodiments, such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle size and / or droplet size in the range of about 0.1 pm to about 200 pm, and may further contain one or more additional ingredients as described herein.

[0141] General considerations in the formulation and / or manufacture of pharmaceutical preparations can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005.

[0142] In some embodiments, a therapeutically effective amount of the tLCNP formulation described herein is delivered to a patient and / or animal before, simultaneously with, and / or after diagnosis of the disease. In some embodiments, a therapeutic amount of the composition of the present invention is delivered to a patient and / or animal before, simultaneously with, and / or after the onset of disease symptoms. In some embodiments, the amount of the tLCNP formulation described herein is sufficient to treat, reduce, improve, alleviate, or relieve one or more symptoms or features of the disease, delay its onset, inhibit its progression, reduce its severity, and / or reduce its incidence. In some embodiments, the amount of the tLCNP formulation described herein is sufficient to elicit a detectable immune response in a subject. In some embodiments, the amount of the tLCNP formulation described herein is sufficient to elicit a detectable T-cell response in a subject. In some embodiments, the amount of the tLCNP formulation described herein is sufficient to elicit a detectable antibody and T-cell response in a subject. In some embodiments, the provided nanoparticles have the advantage that they can elicit an effective response with antigens at much lower concentrations than those required for conventional controls.

[0143] According to the methods described herein, the composition can be administered in any amount and via any route of administration that is therapeutically effective. The exact amount required will vary from subject to subject, depending on the subject’s species, age and general condition, severity of infection, specific composition, route of administration, mode of activity, etc. Compositions containing the tLCNP formulation described herein are typically formulated in unit dosage form for ease of administration and uniform dosing. However, it should be understood that the total daily dose of the composition will be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective (or prophylacticly effective) dose level for any particular subject or organism will depend on a variety of factors, including the condition being treated and its severity; the activity of the specific active ingredient used; the specific composition used; the subject’s age, weight, general health condition, sex, and diet; the timing, route of administration, and excretion rate of the specific active ingredient used; the duration of treatment; drugs used in combination with or concurrently with the specific active ingredient used; and similar factors well known in the medical field.

[0144] In some embodiments, the pharmaceutical composition is administered via multiple routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intravenous, transdermal, intradermal, rectal, vaginal, intraperitoneal, local (e.g., via powder, ointment, cream, and / or drops), mucosal, nasal, buccal, intestinal, and sublingual; via endotracheal instillation, bronchial instillation, and / or inhalation; and / or as an oral spray, nasal spray, and / or aerosol. Specifically, the routes of administration considered are oral, intravenous, intramuscular, and / or subcutaneous injection. In some embodiments, the nanoparticles are administered parenterally. In some embodiments, the nanoparticles are administered intravenously. In some embodiments, the nanoparticles are administered orally.

[0145] Generally, the most appropriate route of administration will depend on a variety of factors, including the nature of the tLCNP formulation described herein (e.g., its stability in the gastrointestinal environment), the condition of the subject (e.g., whether the subject can tolerate oral administration), etc.

[0146] In some embodiments, the lyophilized RNA-LNPs of the present invention encompass a “therapeutic mixture” comprising a group of lyophilized RNA-LNP formulations described herein. In some embodiments, all lyophilized RNA-LNP formulations described herein within a nanoparticle group comprise a single type of cargo (which may be one type of RNA or a mixture of two or more RNAs). In some embodiments, different nanoparticles within a nanoparticle group comprise two or more different RNA cargoes.

[0147] In some embodiments, the pharmaceutical composition is formulated for use in vaccination (e.g., human vaccination).

[0148] Another aspect of the invention provides a kit comprising (a) lyophilized RNA-LNP or pharmaceutical composition as described herein; and (b) an aqueous reconstitution solution (e.g., sterile water for injection).

[0149] In some embodiments, the lyophilized RNA-LNP package is used for storage at 2°C to 8°C, and the aqueous reconstitution solution package is used for storage at room temperature (RT or 20°C to 25°C).

[0150] Another aspect of the present invention provides a method for reconstituted lyophilized RNA-LNPs described herein, the method comprising adding an aqueous liquid to the lyophilized RNA-LNPs.

[0151] In some implementations, the aqueous liquid is sterile water for injection (WFI).

[0152] Another aspect of the invention provides a method for immunizing a mammal, the method comprising administering to a mammal lyophilized mRNA-LNP (RNA-lipid nanoparticles) reconstituted in sterile water for injection (WFI) as described herein, wherein the RNA encodes an antigen vaccine.

[0153] In some implementations, the mammal is human (e.g., human children aged 6 months to 4 to 5 years; human children aged 5 or 6 to 11 years; human children aged 12 to 17 years; or human adults aged 18 years and older).

[0154] In some implementations, mammals are non-human animals, non-human mammals, or non-human primates (e.g., livestock such as cattle, oxen or bulls, sheep, goats, horses, pigs, camels, etc.; pets such as cats or dogs; or rodents such as rabbits, mice, rats, hamsters, guinea pigs, etc.).

[0155] In some implementations, the reconstituted lyophilized RNA-LNP is intravenously injected into mammals.

[0156] In some implementations, the RNA is mRNA encoding the spike protein (e.g., S-2P protein) or an antigenic fragment of the SARS-CoV-2 virus (e.g., the Omecron variant of the SARS-CoV-2 virus).

[0157] Based on the general aspects of the invention described herein, the following embodiments further illustrate specific aspects of the invention, and these embodiments are not limiting in any way. Any specific implementation described in the embodiments is also a specific implementation of the invention and may / is intended to be combined with other aspects of the invention or other implementations described above.

[0158] Example The following examples provide, in particular, proof-of-concept examples demonstrating the advantageous pharmaceutical and biological properties of the mRNA vaccine products of the present invention (e.g., lyophilized mRNA-LNPs) prepared using the methods of the present invention. A series of formulations with different compositions were then screened to elucidate the key formulation characteristics required to obtain the subject mRNA products with acceptable pharmaceutical and biological properties.

[0159] The examples provided herein are generally for illustrative purposes only and are not restrictive. However, the detailed conditions and examples described in the embodiments are specific implementations of the invention and are generally applicable to any implementation or aspect of the invention described in other parts of the specification.

[0160] Example 1: Preparation of S-2P mRNA To prepare mRNA encoding the SARS-CoV-2 S-2P protein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein was amplified in bacterial culture according to a standard plasmid amplification protocol. The amplification product was then extracted and purified. Next, the plasmid was linearized, followed by purification by chromatography and ethanol precipitation.

[0161] Using purified linearized plasmid DNA as a template, SARS-CoV-2 spike glycoprotein mRNA was synthesized in vitro via enzymatic synthesis, and the purified mRNA was stored at -80°C.

[0162] Example 2 Preparation of mRNA-LNP intermediate A liquid dispersion of mRNA encapsulated in lipid nanoparticles (LNPs) was formulated by rapidly mixing the ethanol and aqueous phases using a microfluidic device. The aqueous phase contained an amount of S-2P mRNA dissolved in 50 mM citrate buffer (pH 6.0). The ethanol phase consisted of ionizable lipids (Immorna), cholesterol (Jiangsu Southeast Nanomaterials Co., Ltd.), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC) (Jiangsu Southeast Nanomaterials Co., Ltd.), and 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000) (SINOPEG). Unless otherwise specified, these four lipid components were mixed in a molar ratio of 40:48:10:2.0 (ionizable lipids:cholesterol:DSPC:DMG-PEG2000).

[0163] The resulting mRNA-LNP liquid dispersion was then purified in citrate buffer (pH 6.0) containing a protective agent such as sucrose, and characterized in terms of particle size and polydispersity index (PDI), as well as mRNA concentration, encapsulation efficiency, and purity. Finally, the pH of the mRNA-LNP liquid dispersion was adjusted to 7.2, and then diluted to the target concentration to obtain the mRNA-LNP intermediate (stored at 2°C to 8°C).

[0164] Example 3 Preparation of lyophilized mRNA-LNP The mRNA-LNP intermediate of Example 2 was freeze-dried according to a freeze-drying process including pre-freezing, primary drying, and secondary drying. Thus, a freeze-dried form of mRNA-LNP (i.e., freeze-dried mRNA-LNP) with extremely low moisture content (<3%) was obtained.

[0165] Like many other products in lyophilized form, the resulting lyophilized mRNA-LNP can be stored at 2°C to 8°C for extended periods without impairing the bioactivity of the mRNA-LNP product.

[0166] Example 4: Preparation and Characterization of the Final mRNA-LNP Product Before applying the mRNA-LNP product, a reconstituted solution (e.g., sterile water for injection) was added to the lyophilized mRNA-LNP of Example 3, and then mixed at room temperature to obtain the final mRNA-LNP product, which is a uniform liquid dispersion.

[0167] Specifically, after adding the reconstituted solution to the lyophilized S-2 mRNA-LNP, a uniform mRNA-LNP liquid dispersion was obtained within seconds, with an appearance consistent with the mRNA-LNP intermediate control (i.e., the liquid dispersion obtained before lyophilization). The reconstituted mRNA-LNP was a milky white, uniform dispersion with no visible particles.

[0168] Furthermore, mRNA concentrations of up to at least 60 μg / mL (mRNA equivalent) have proven feasible for the subject mRNA-LNP formulation. Such high concentrations should be sufficient for many mRNA products, such as mRNA vaccine products.

[0169] Aside from appearance, other key drug characterizations were comparable between the two mRNA-LNP assays, including polydispersity index (PDI) and mRNA purity. See Table 1 below for data.

[0170] Table 1. Representative drug characterization of S-2P mRNA-LNP liquid assay (prepared with and without lyophilization process)

[0171] * The No. 1 formulation (S-2P mRNA-LNP liquid dispersant intermediate) was lyophilized and then reconstituted to obtain the No. 2 formulation (i.e., the S-2P mRNA-LNP product). In one batch, the lyophilized mRNA-LNP formulation of the present invention exhibited a larger particle size after reconstitution compared to a control mRNA-LNP liquid dispersant. However, the particle size was still well below 250 nm and was therefore considered suitable for the intended application.

[0172] These data indicate that the lyophilization process does not negatively affect the drug characterization of mRNA-LNP products.

[0173] Although not necessarily limited, in the embodiments described herein, unless otherwise stated, the resulting mRNA-LNP final product contains approximately 60 μg / mL S-2P mRNA.

[0174] Example 5 In vitro transfection This experiment showed that, at the same dose (4 μg), the transfection efficiency of the subject-specific lyophilized S-2P mRNA-LNP product and the control was comparable in both BHK and Vero E6 cell lines.

[0175] In short, in this experiment, the amount of SARS-CoV-2 spike glycoprotein expressed in transfected cells was detected by Western blotting at 24, 48, and 72 hours post-transfection (see [link to study]). Figure 1 Based on the comparable expression levels of the S-2P protein, it can be concluded that the mRNA-LNP product described herein is at least as effective as the mRNA-LNP intermediate / control that remains in liquid dispersible form without a lyophilization process.

[0176] Specifically, for vaccine transfection in SARS-CoV-2 susceptible Vero E6 and BHK-21 cells, the cells were loaded at a rate of approximately 3 × 10⁻⁶ cells / year.5 Cells were seeded at a density of 10 cells / well in 6-well plates. Equal volumes (4 μg) of the theme S-2P mRNA-LNP product prepared by reconstituted and lyophilized theme mRNA-LNP and the S-2P mRNA-LNP liquid dispersant control (prepared according to standard manufacturing process, i.e., not lyophilized) were transfected into cells, respectively.

[0177] Following transfection, the spike glycoprotein of SARS-CoV-2 was collected from the cell supernatant using TCA for the following assays. At 24, 48, and 72 hours post-transfection, 55 μL of 100% TCA was added to 0.5 mL of cell supernatant, and the mixture was incubated on ice for 30 min, followed by occasional vortexing and centrifugation for 30 min (13,000 rpm, 4 °C). The supernatant was aspirated, the precipitate was collected, and washed three times with 1 mL of ethanol. Then, 2.5 × SDS was added, followed by boiling at 95 °C for 12 min, separation on a 6% SDS-PAGE gel, and transfer to a nitrocellulose filter membrane.

[0178] After blocking with 5% BSA, the membrane was blotted with primary antibody (1:1000) (SARS-CoV-2 (2019-nCoV) spike rabbit PAb, 40592-T62, Sino Biological), followed by incubation with secondary antibody conjugated with horseradish peroxidase (HRP) (1:10,000) (IgG(H+L) (HRP-labeled goat anti-rabbit IgG(H+L)), Beyotime), and visualized using a chemiluminescent reagent (chemiluminescent HRP substrate, WBKLS0500, Millipore).

[0179] Furthermore, in the formulation screening study (see Example 8 below), the in vitro transfection efficiency of the Theme S-2P mRNA-LNP assay (prepared by reconstitution of lyophilized mRNA-LNP) comprising different lipid compositions was evaluated in both BHK-21 and Vero E6 cell lines. In short, 3 × 10 5 One Vero E6 or BHK-21 cell per well was seeded in a 6-well plate. 4 μg of the subject S-2P mRNA-LNP product (prepared by reconstitution of lyophilized mRNA-LNP) or the S-2P mRNA-LNP liquid dispersion control (not lyophilized) was transfected into the cells. Forty-eight hours post-transfection, RBD expression in the cell culture supernatant was quantified using a commercial SARS-CoV-2 (2019-nCoV) spike RBD ELISA kit (KIT40592, Sinocare) according to the manufacturer's instructions.

[0180] Specifically, the supernatant was diluted 200-fold. The final concentration of RBD was calculated based on a linear standard curve of absorbance at 450 nm. In short, the detection wells were pre-coated with a monoclonal antibody specific for the spike RBD protein. After incubating with the sample or standard at room temperature for two hours, samples not bound to the immobilized antibody were removed by washing. The detection antibody was then added to the wells and incubated at RT for one hour. After washing, the substrate solution was added to each well while protecting from light. After 20 minutes, stop solution was added to each well, and absorbance at 450 nm was measured.

[0181] Example 6: In vivo evaluation of reconstituted lyophilized S-2P mRNA-LNP product In vivo studies were conducted to evaluate BALB / c mice and cynomolgus monkeys (cynomolgus macaques). Macaca fascicularis Antibody response following vaccination with S-2P mRNA encapsulated with LNPs encoding the SARS-CoV-2 spike glycoprotein. In these studies, mice and cynomolgus monkeys received an initial injection followed by a booster injection three weeks later (see [link to study]). Figure 2 The vaccine test items administered were mRNA-LNP liquid dispersion control or reconstituted lyophilized mRNA-LNP product (prepared by lyophilizing mRNA-LNP intermediates and then reconstituted).

[0182] Because antibody titers are highly correlated with the protective effect and durability of vaccination, they are used as efficacy readings in this embodiment. Figure 3 and Figure 4 The titer is presented in the form of geometric mean titer (GMT).

[0183] like Figure 3 (BALB / c mice) and Figure 4 As shown in the (cynomolgus monkey) studies, the Subject S-2P mRNA-LNP product and the liquid dispersion control induced similar antibody responses on days 13 and 20 following the first vaccination (primary injection). For both mRNA vaccines, antibody titers increased significantly after two immunizations. Between these two, the Subject S-2P mRNA-LNP vaccine product appeared to elicit comparable (mice) or even higher (cynomolgus monkey) antibody responses. These data demonstrate that the Subject mRNA-LNP product is at least as effective as the mRNA-LNP liquid dispersion control.

[0184] More specifically, in order to evaluate the efficacy of the reconstituted lyophilized mRNA-LNP product of the present invention, animal (mouse) studies were conducted at the Yangtze River Delta Research Institute of Tsinghua University (Zhejiang, China).

[0185] BALB / c mice (6 to 8 weeks old) were randomly divided into 4 groups (n=4). On day 0 (initial injection) and day 21 (booster injection), the three groups of mice were administered intramuscular injections of lyophilized S-2P mRNA-LNP vaccine (8 μg mRNA), LNP-encapsulated S-2P mRNA vaccine (5 μg mRNA), and buffered solvent, respectively. im Immunization. Serum from mice was collected before the first vaccination and on days 13, 20, 28, and 35. All collected samples were frozen according to standard protocol.

[0186] In another experiment, animal (cynomolgus monkey) studies were conducted at Shanghai Hekai Biotechnology Co., Ltd. The cynomolgus monkeys were divided into one group (n=1). On day 0 (initial injection) and day 21 (booster injection), both groups of cynomolgus monkeys were administered intramuscularly with either lyophilized S-2P mRNA-LNP vaccine (30 μg mRNA) or LNP-encapsulated S-2P mRNA vaccine (30 μg mRNA). im Immunization. Serum from monkeys was collected before the first vaccination and on days 13, 20, 28, and 35. All collected samples were frozen according to standard protocol.

[0187] Example 7: Quantification of antibody binding titer against SARS-CoV-2 spike protein by ELISA assay The antibody binding titer against the SARS-CoV-2 spike protein (RBD, His tag) (GenScript Z03483) was quantified by enzyme-linked immunosorbent assay (ELISA).

[0188] In short, SARS-CoV-2 spike protein (RBD, His tag) diluted at 0.5 μg / mL in carbonate-bicarbonate buffer was pre-coated overnight on 96-well clear polystyrene microplates (Corning) 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) at 37°C for 1 hour. Serum samples were serially diluted 2-fold in blocking buffer, transferred to the plates, and incubated at 37°C for 1 hour. After washing, the plates were incubated with HRP-conjugated rabbit anti-mouse IgGH+L (Abcam, ab6728) at 37°C for 1 hour. The plates were washed and incubated with TMB single-component substrate solution (Solarbio, PR1200) at 37°C for 7 minutes, and the reaction was terminated with ELISA stop solution (Solarbio, C1058). The absorbance was read at 450 nm using a microplate reader (VARIOSKAN LUX, ThermoFisher), and the ELISA titer was determined using nonlinear 4-parameter variable slope analysis in GraphPad Prism 8 software.

[0189] Example 8: Screening LNP compositions suitable for lyophilized mRNA-LNP products The drug characterization and biological properties of LNP-encapsulated mRNA formulations are at least in part influenced by the chemical structure of the ionizable lipids, the ratios between the lipid components that make up the LNP, and other formulation and manufacturing process parameters.

[0190] Based on the accumulation of lyophilized mRNA-LNP products, two formulation variables—i) ionizable lipid structure and ii) the ratio of lipid components—were systematically evaluated in a series of experiments to determine the range of LNP compositions suitable for the present invention as described herein. It should be emphasized that all formulations evaluated in the following screening studies were prepared according to standard protocols used for comparison, without further optimization of formulation or manufacturing process parameters.

[0191] In the above embodiments, a unique ionizable lipid called "lipid 11" was used (see [link to documentation]). Figure 5 The chemical structures of the reconstituted lyophilized mRNA-LNP products (e.g., prepared from lyophilized mRNA-LNP) were used to demonstrate the chemical structures of the reconstituted lyophilized mRNA-LNP products. To assess the impact of ionizable lipid structures on the corresponding drug profiles, several other ionizable lipids with significantly different chemical structures (referred to as "lipoprotein 10, Dlin-MC3-DMA, lipid 2, and lipid 4") were selected in parallel with lipid 11 (see also their chemical structures in [reference needed]). Figure 5The chemical structures in the LNP were used together with three other lipid components of LNP (DMG-PEG, DSPC, and cholesterol) to prepare the S-2P mRNA-LNP intermediate according to the above scheme.

[0192] Although these five LNP formulations are composed of different ionizable lipids, they use the same lipid molar ratio as the formulations mentioned above, namely 40:48:10:2.0 (ionizable lipids: cholesterol: DSPC: DMG-PEG2000). See Table 2.

[0193] Table 2. Formulation parameters of S-2P mRNA-LNP products prepared using different ionizable lipids

[0194] The generated mRNA-LNP intermediate assay appeared as a uniform, milky-white liquid dispersion and was used as a head-to-head control for mRNA-LNP liquid dispersion. Additionally, a portion of the generated mRNA-LNP intermediate assay was lyophilized, stored at 2°C to 8°C (if desired), and then reconstituted in aqueous solution to generate the subject mRNA-LNP product, which was then used for pharmaceutical or biological evaluation.

[0195] First, the drug characterization (Table 3) and in vitro transfection efficiency of the S-2P mRNA-LNP products prepared using these five ionizable lipids were compared. Figure 6 Then, the stability characteristics of the first two mRNA-LNP (lyophilized) test items (formulation 1 and formulation 2) were evaluated at 2°C to 8°C (see Table 4).

[0196] Table 3. Physical properties of S-2P mRNA-LNP products prepared using different ionizable lipids.

[0197]

[0198] Note: i) EE = mRNA encapsulation efficiency ii) The mRNA-LNP liquid dispersant control was lyophilized and then reconstituted to obtain the mRNA-LNP product. Table 4. Storage conditions for 4 weeks at 2°C to 8°C The S-2P mRNA-LNP product prepared for the mRNA-LNP (lyophilized) assay contains the first two ionizable lipids (lipoprotein 11 and lipid 10). Drug characterization and in vitro transfection efficiency compared with freshly prepared S-2P mRNA-LNP products

[0199] As shown in Table 3, the choice of ionizable lipids affects the particle size of the mRNA-LNP products of this invention at the used / fixed lipid molar ratio. Formulations prepared using the applicant's proprietary ionizable lipids (lipoproteins 11, 10, 2, and 4) appear to have smaller particle sizes and fall within acceptable ranges for the intended application after, for example, intramuscular administration (even without further formulation and process parameter optimization). In contrast, similar formulations prepared using the more typical ionizable lipid Dlin-MC3-DMA have larger particle sizes.

[0200] Furthermore, the in vitro transfection efficiency of these five mRNA-LNP assays was compared in the BHK-21 cell line at equal doses (4 μg), and the corresponding protein expression levels were determined by Western blotting (WB). Figure 6 As shown, all four mRNA-LNP products containing the applicant's proprietary ionizable lipids exhibited better performance than Dlin-MC3-DMA ( Figure 6 Lane 3) Better transfection efficiency. Notably, the two formulations prepared with lipid 11 and lipid 10 showed significantly better expression efficiency than the other three ionizable lipids, most likely due to the presence of unsaturated double bonds in lipid 11 and lipid 10.

[0201] It is also expected that the structure of ionizable lipids will affect the stability characteristics of the resulting mRNA-LNP assays in lyophilized form. As shown in Table 4, the first two ionizable lipids (i.e., lipid 11 and lipid 10) appear to produce fairly stable mRNA-LNP (lyophilized) assays at 2°C to 8°C, as evidenced by their substantially unchanged drug properties and in vitro transfection efficiency upon reconstitution after storage at 2°C to 8°C for 4 weeks.

[0202] Overall, both ionizable lipids (lipoprotein 11 and lipid 10) are ideal candidates for constructing thematic lyophilized mRNA-LNP and reconstituted mRNA-LNP final products.

[0203] The ionizable lipids with this structure appear to produce an mRNA-LNP liquid dispersion (intermediate) with good pharmaceutical characterization, which, upon lyophilization, yields a stable lyophilized mRNA-LNP vaccine at 2°C to 8°C. Once reconstituted, the latter forms the final mRNA-LNP product, which exhibits good pharmaceutical characterization and unimpaired biological activity compared to the liquid dispersion control.

[0204] This data indicates that ionizable lipids with the general formula (I) , Formula (I) R1 and R2 are independently C1 to C3 alkyl groups; R3 is a C1 to C4 alkyl group; R4 and R5 are independently C1 to C5 alkyl groups. 10 Alkyl groups; Q1, Q2, and Q3 are each independently -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-, -SS-; L is -(CH2). 0-2 -CH<or R6 and R7 are independently C1 to C 10 Alkyl or C1 to C 10 Alkenyl; A1 and A2 are each independently bonded, -C-, -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-, -SS-; R8 and R9 are independently C1 to C 10 Alkyl or C1 to C 10 Alkenyl groups; these combinations are best suited for developing the lyophilized mRNA-LNP products described in this article.

[0205] Example 9: Mole fraction of lipid components constituting LNP LNPs typically comprise four key lipid-based components: 1) ionizable lipids, 2) phospholipids (e.g., DSPC), 3) cholesterol, and 4) PEGylated lipids (e.g., DMG-PEG2000). Each of these components and their relative proportions play a crucial role in the construction of the LNP. Therefore, for the development of the subject-specific lyophilized mRNA-LNP formulations and end products, it is beneficial to optimize mRNA-LNPs composed of different proportions of lipids in order to identify the lipid compositions best suited for the mRNA-LNP products described herein. This embodiment provides data illustrating some exemplary (non-limiting) lipid compositions more suitable for the mRNA-LNP products described herein.

[0206] To determine the lipid ratio range, the amount of ionizable lipids in the mRNA-LNP formulation was adjusted between 25 mol% and 70 mol%, with the cholesterol content varying accordingly, while other lipid components remained constant.

[0207] Under this experimental design, the effect of the molar fraction of lipid components on the formation and drug characterization of the subject mRNA-LNP product was assessed independently or jointly.

[0208] The readings in Table 5 indicate that, considering both ionizable lipid structures tested, the drug characterization of the resulting mRNA-LNP final product (in terms of particle size and mRNA encapsulation efficiency) appears to be poor, or negatively affected by the lyophilization process, when the ionizable lipid fraction reaches 70 mol% or decreases to 25 mol%. Therefore, the ionizable lipid fraction is ideally between approximately 25 mol% and 70 mol%.

[0209] Table 5. Drug characterization of S-2P mRNA-LNP products and liquid dispersant controls prepared using different molar fractions of ionizable lipids.

[0210] EE = mRNA encapsulation efficiency; N / P = 7 (mol / mol) In addition to drug characterization, the in vitro transfection efficiency of these mRNA-LNP assays was evaluated in BHK-21 and Vero E6 cell lines at equal doses (4 μg). As shown in Table 6, high transfection efficiency was achieved when the molar ratio of ionizable lipids was at least 40 mol% to 55 mol% for both tested ionizable lipid structures.

[0211] Table 6. In vitro transfection of thematic S-2P mRNA-LNP products prepared with different lipid ratios in BHK-21 or Vero E6 cell lines, presented as RBD expression levels (as determined by ELISA).

[0212] N / P ratio = 7 (mol / mol) Based on experimental evaluation, Table 7 summarizes some of the best LNP compositions suitable for constructing and stabilizing the subject-specific lyophilized mRNA-LNP, as well as the pharmaceutical and biological characteristics of the reconstituted mRNA-LNP products.

[0213] Table 7. Formulation compositions and parameters suitable for lyophilized mRNA-LNP products

Claims

1. A lyophilized RNA-LNP (RNA-lipid nanoparticle, e.g., mRNA-LNP), wherein the RNA-lipid nanoparticle comprises RNA (e.g., mRNA) encapsulated in a lipid nanoparticle (LNP), and wherein the LNP comprises an ionizable lipid having the structure of formula (I): Formula (I), in, R1 and R2 are independently C1-3 alkyl groups; R3 is C 1-4 alkyl; R4 and R5 are independently C1- 10 alkyl; Q1, Q2 and Q3 are each independently -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O- or -SS-; L stands for -(CH2) 0-2 -CH<or (For example, R4 and R5 are connected to L via -O-). R6 and R7 are independently C1- 10 Alkyl or C1- 10 alkenyl; A1 and A2 are each independently a bond, -O-, -S-, -OC(O)-, -C(O)-O-, -OC(S)-, -C(S)-O-, or -SS-, and R8 and R9 are independently C1- 10 Alkyl or C1- 10 alkenyl; Optionally, R6 contains at least one (e.g., 1) double bond, R7 contains at least one (e.g., 1) double bond, R8 contains at least one (e.g., 1 or 2) double bond, and / or R9 contains at least one (e.g., 1 or 2) double bond.

2. The lyophilized RNA-LNP according to claim 1, wherein the LNP further comprises: (1) Sterol lipids (e.g., cholesterol); (2) Phospholipids (e.g., DSPC), and (3) Polyethylene glycol-modified lipids (e.g., DMG-PEG2000).

3. The freeze-dried RNA-LNP according to claim 1 or 2, wherein the moisture content of the freeze-dried RNA-LNP is < about 5% (e.g., < about 3%).

4. The lyophilized RNA-LNP according to any one of claims 1 to 3, wherein the lyophilized RNA-LNP is stable under storage conditions of 2°C to 8°C (e.g., after 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks, the loss of biological activity (such as transfection efficiency) of the RNA-LNP does not exceed 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%).

5. The lyophilized RNA-LNP according to any one of claims 1 to 4, wherein when the lyophilized RNA-LNP is reconstituted in a reconstitution solution (e.g., sterile water for injection or WFI), the lyophilized RNA-LNP is substantially free of visible particles.

6. The lyophilized RNA-LNP according to claim 5, wherein the RNA concentration of the reconstituted lyophilized RNA-LNP is at least about 60 μg / mL (RNA equivalent).

7. The lyophilized RNA-LNP according to any one of claims 1 to 6, wherein, after reconstitution, substantially all (e.g., >80%, 85%, 90%, 90%, 95% of all particles) of particles have a size <250 nm (e.g., <200 nm, <190 nm, <180 nm, <170 nm, <160 nm or <150 nm).

8. The lyophilized RNA-LNP according to any one of claims 1 to 7, wherein the combined molar ratio of the ionizable lipid and the cholesterol is about 85% to 90%, for example about 88%.

9. The lyophilized RNA-LNP according to any one of claims 1 to 8, wherein the molar ratio of said ionizable lipid is about 25 mol% to 70 mol%, about 35 mol% to 60 mol%, about 35 mol% to 55 mol%, or about 40 mol% to 55 mol%.

10. The lyophilized RNA-LNP according to any one of claims 1 to 9, wherein the molar ratio of the ionizable lipid, the cholesterol, the phospholipid and the polyethylene glycol-modified lipid is about 40:48:10:2.

0.

11. The lyophilized RNA-LNP according to any one of claims 1 to 10, wherein R1 and R2 are both -Me.

12. The lyophilized RNA-LNP according to claim 11, wherein Q1 is -OC(O)- or -C(O)-O-, and is linked to L by -O- or -C(O)-.

13. The lyophilized RNA-LNP according to claim 12, wherein L is -CH2-CH<; R4 and R5 are both -(CH2)7-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 is C9-alkenyl; R7 is C 8-9 Alladienyl; A1, A2, and R8 are all bonds; and R9 is C. 7-8 Alkenyl group.

14. The lyophilized RNA-LNP according to claim 13, wherein the ionizable lipid has the following structure: (Lipid No. 10).

15. The lyophilized RNA-LNP according to claim 12, wherein L is -CH<; R4 and R5 are both -(CH2)8-; Q2 and Q3 are both -C(=O)-O- linked to R4 and R5 via -C(=O)-; R6 and R7 are both C6-alkenyl; A1 and A2 are both -O-; and R8 and R9 are both -(CH2)6-.

16. The lyophilized RNA-LNP according to claim 15, wherein the ionizable lipid has the following structure: (Lipid No. 11).

17. The lyophilized RNA-LNP according to any one of claims 1 to 16, wherein the RNA: (a) is a self-replicating RNA (e.g., mRNA) or a non-self-replicating RNA (e.g., mRNA), and / or (b) Encoding immunogenic antigens (e.g., one or more immunogenic antigens derived from the same or different proteins).

18. The lyophilized RNA-LNP of claim 17, wherein the immunogenic antigen comprises a viral protein or a cancer antigen.

19. The freeze-dried RNA-LNP of claim 18, wherein the viral protein is the spike protein (e.g., S-2P protein) of SARS-CoV-2 virus (e.g., the Omecron variant of said SARS-CoV-2 virus) or an antigenic fragment thereof.

20. A method for producing lyophilized RNA-LNP according to any one of claims 1 to 19, the method comprising lyophilizing a liquid dispersant comprising the RNA-lipid nanoparticles.

21. The method of claim 20, wherein the liquid dispersant is formulated by mixing an organic phase (e.g., an ethanol phase) with an aqueous phase using a microfluidic device, wherein: (1) The organic phase (e.g., the ethanol phase) comprises: ionizable lipids, cholesterol, phospholipids (e.g., 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC)) and polyethylene glycol-modified lipids (e.g., 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000)); and (2) The aqueous phase contains RNA dissolved in an aqueous buffer (such as 50 mM citrate buffer (pH 6.0)).

22. The method of claim 20 or 21, further comprising purifying the liquid dispersant in the aqueous buffer solution, optionally containing a protectant (e.g., sucrose), prior to lyophilization, and adjusting the pH to a predetermined level (e.g., pH 7.2, if necessary).

23. The method of claim 22, further comprising adjusting the concentration of the liquid dispersant prior to freeze-drying and / or storing the liquid dispersant at 2°C to 8°C.

24. The method according to any one of claims 20 to 23, wherein the liquid dispersant is freeze-dried by a freeze-drying process comprising pre-freezing, primary drying, and secondary drying.

25. A lyophilized RNA-LNP produced by the method according to any one of claims 20 to 24.

26. A pharmaceutical composition comprising lyophilized RNA-LNP (e.g., mRNA-LNP) according to any one of claims 1 to 19 and 25 and a pharmaceutically acceptable excipient.

27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is formulated for use in vaccination (e.g., human vaccination).

28. A kit comprising (a) lyophilized RNA-LNP according to any one of claims 1 to 19 and 25, or a pharmaceutical composition according to claim 27 or 28; and (b) an aqueous reconstitution solution (e.g., sterile water for injection).

29. The kit of claim 28, wherein the lyophilized RNA-LNP package is for storage at 2°C to 8°C, and wherein the aqueous reconstitution solution package is for storage at room temperature (RT or 20°C to 25°C).

30. A method for reconstituted lyophilized RNA-LNP according to any one of claims 1 to 19 and 25, the method comprising adding an aqueous liquid to the lyophilized RNA-LNP.

31. The method of claim 30, wherein the aqueous liquid is sterile water for injection (WFI).

32. A method of immunizing a mammal, the method comprising administering to the mammal lyophilized mRNA-LNP (RNA-lipid nanoparticles) reconstituted in sterile water for injection (WFI) according to any one of claims 1 to 19 and 25, wherein the RNA encodes an antigen vaccine.

33. The method of claim 32, wherein the mammal is a human (e.g., a human child aged 6 months to about 4 to 5 years; a human child aged 5 or 6 to 11 years; a human child aged 12 to 17 years; or a human adult aged 18 years and older).

34. The method of claim 32 or 33, wherein the RNA is mRNA encoding a spike protein (e.g., S-2P protein) or an antigenic fragment of the SARS-CoV-2 virus (e.g., an Omeprone variant of the SARS-CoV-2 virus).

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