Methods and compositions for freeze-drying lipid nanoparticles

Abstract

The present invention provides lyophilized nucleic acid lipid nanoparticles (NALNPs) comprising (a) lipid nanoparticles comprising a nucleic acid, and (b) a lyophilization buffer comprising a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent, as well as methods for preparing the same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S.C. § 119(e) to U.S. patent application Ser. No. 18 / 479,482, filed December 12, 2022. [Background technology]

[0002] The short production time of mRNA vaccines makes them ideal for rapid response to emerging threats such as the COVID-2019 virus. Furthermore, nucleic acid-based vaccines offer advantages over conventional vaccines in terms of safety and efficacy. Messenger RNA ("mRNA") vaccines are competitive with DNA-based vaccines, which must cross the nuclear membrane to function and risk integration into the host genome. Without a delivery system, mRNA vaccines require a carrier because they are subject to in vivo degradation by exonucleases and endonucleases. Currently, lipid nanoparticles (LNPs) are one of the most commonly used vectors for in vivo RNA delivery. Lipid nanoparticles or LNPs generally consist of a lipid or aqueous core surrounded by a lipid bilayer shell made of a combination of different lipids, each performing a specific function.

[0003] Current mRNA vaccines must be kept frozen at low temperatures for storage. This requirement limits the distribution of the vaccines. Therefore, there is still a need for a suitable freeze-drying protocol to keep mRNA vaccines in a stable and effective form. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2020252589 Brochure [Patent Document 2] International Publication No. 2021000041 Brochure [Patent Document 3] U.S. Patent No. 5,753,613 [Patent Document 4] U.S. Patent No. 6,734,171 [Patent Document 5] U.S. Patent No. 9,758,795 [Patent Document 6] U.S. Patent No. 9,943,846 [Patent Document 7] U.S. Patent No. 10,159,652 [Patent Document 8] International Publication No. 2017117647 Brochure [Patent Document 9] U.S. Patent No. 10,076,730 [Patent Document 10] International Publication No. 2018006166 Brochure [Patent Document 11] U.S. Patent No. 9,943,846 [Patent Document 12] U.S. Patent No. 10,076,730 [Patent Document 13] U.S. Patent Application No. 20040262223 [Patent Document 14] International Publication No. 2020 / 252589 Brochure [Patent Document 15] International Publication No. 2021 / 000041 Brochure [Patent Document 16] U.S. Patent No. 7,425,337 [Non-patent literature]

[0005] [Non-Patent Document 1] Zhang, Sh et al., Chemical Eng. J. 144(2): 324-328 (2008) [Non-patent document 2] Jeffs, L.B. et al., Pharm. Res., 22(3): 362-372 (2005) Summary of the Invention [Means for solving the problem]

[0006] According to one embodiment, (a) lipid nanoparticles containing nucleic acids, and (b) a lyophilization buffer solution containing a sugar and a lyophilization reagent selected from a sugar-mimetic oligomer / polymer, an amphiphilic thermoresponsive polymer, an ethylene glycol-mimetic polymer, a hydrophilic monomer, and a hydrophilic polymer; A lyophilized nucleic acid lipid nanoparticle (NALNP) comprising:

[0007] In some embodiments, the glycomimetic oligomer / polymer is n-octanoylsucrose, cyclodextrin, beta-cyclodextrin polymer, dextran, trehalose, sorbitol-core carboxy-terminated PEG, Betadex™ sulfobutyl ether sodium, or 2-hydroxypropyl-beta-cyclodextrin.

[0008] In other embodiments, the amphiphilic thermoresponsive polymer is poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide).

[0009] In other embodiments, the ethylene glycol-mimetic polymer is a 6-arm branched PEG, a 5-arm branched PEG, a 3-arm branched PEG, trimethylpropane ethoxylate, polyethylene glycol, poloxamer 407, an amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol).

[0010] In yet other embodiments, the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), an amino acid, or L-arginine.

[0011] In embodiments of the present invention, the mass / mass ratio of lyophilized reagent to sugar is 1:8 or 1:4. In embodiments, the pharmaceutically acceptable diluent is selected from Tris, sodium acetate, sodium citrate, dextrose, and saline solutions. In embodiments, the NALNP has a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000. In embodiments, the sugar is sucrose. In many embodiments, the NALNP is in anhydrous form.

[0012] According to one embodiment, (a) mixing a nucleic acid with a lipid mixture solution containing an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles; (b) combining the lipid nanoparticles obtained in (a) with a lyophilization buffer containing a sugar and a lyophilization reagent selected from the group consisting of polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-beta-cyclodextrin, N,N-dimethylacrylamide, poly(N,N-diethylacrylamide), poly(2-ethyl-2-oxazoline), glycerol ethoxylate, amine-terminated 4-arm PEG, 6-arm branched PEG, 5-arm branched PEG, and sorbitol-core-containing carboxy-terminated PEG, and a pharmaceutically acceptable diluent; (c) lyophilizing the combination of lipid nanoparticles and lyophilization buffer obtained in step (b) to obtain anhydrous lyophilized NALNPs. A method for preparing freeze-dried NALNP is provided, comprising:

[0013] In embodiments, the step of lyophilizing the combination of nanoparticles and lyophilization buffer comprises: (a) freezing a combination of lipid nanoparticles and a lyophilization buffer at −40 to −90° C. for 60 to 400 minutes; (b) drying the combination of lipid nanoparticles and lyophilization buffer at −20 to −40° C. and 30 to 100 mTorr for 700 to 980 minutes; (c) drying the combination of lipid nanoparticles and lyophilization buffer at 4-10°C and 30-100 mTorr for 250-500 minutes; In embodiments, the lyophilization buffer comprises a pharmaceutically acceptable diluent.

[0014] In embodiments, the pharmaceutically acceptable diluent is Tris, sodium acetate, sodium citrate, dextrose, saline, or water. In some embodiments, the lyophilization buffer has a mass / mass ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent of 1:4:40 to 1:8:80. In embodiments, the NALNP has a mass / mass ratio of nucleic acid to lyophilization reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0015] In an embodiment, the sugar is sucrose.

[0016] The features and advantages of the present subject matter will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be recognized, all of the subject matter disclosed and claimed can be modified in various respects without departing from the scope of the claims. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive, the full scope of the subject matter being set forth in the claims. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of an example of a freeze-drying process according to the present invention. [Figure 2] This graph shows the size and polydispersity results of lyophilized LNPs formulated with two different saRNAs (SARS-CoV-2 spike protein-specific A3b or A3p antigens) in VA compositions at N / P-8, reconstituted in 1x phosphate-buffered saline (PBS) after 24 hours (hr) storage at room temperature (RT). [Figure 3]This graph shows the encapsulation efficiency results of lyophilized LNPs made using two different saRNAs (SARS-CoV-2 spike protein-specific A3b or A3p antigens) in VA compositions at N / P-8 using lyophilization buffer (LB) #107 and 110, stored at RT for 24 hours, and reconstituted in 1x PBS. [Figure 4] Figure 1 shows Western blot results of SARS-CoV-2 spike protein expressed in vitro in HEK-293 cells treated with lyophilized LNPs containing PNI 516 and LB#107, 110, 163, 164, 168, 178, 179, 180, 181, 182, 186, and 189, stored at 4°C for 24 hours, and reconstituted with 1 μg / mL saRNA in 1x PBS for 24 hours. [Figure 5] Western blot image showing expression of SARS-CoV-2 spike protein in HEK 293 cells treated with lyophilized PNI 516 LNPs that encapsulated SARS-CoV-2 spike protein-specific A3 saRNA in VB composition (N / P-8), lyophilized, stored at -20°C and 4°C for 1 week, and then reconstituted. [Figure 6] Figure 1 shows the expression of SARS-CoV-2 spike protein in HEK 293 cells as determined by Western blot analysis. LNPs containing PN1 516 and SARS-CoV-2 spike protein-specific A3 saRNA were formulated into a VB composition at N / P-8, lyophilized, stored at RT for 1 week, and reconstituted. [Figure 7] This figure shows the results of Western blot analysis of SARS-CoV-2-specific spike protein expression in HEK 293 cells after treatment with lyophilized LNPs at a saRNA concentration of 0.25 μg / mL. LNPs were formulated with PNI 516 and SARS-CoV-2 spike protein-specific A5 saRNA in a VB composition of N / P-8. After lyophilization, the lyophilized cakes were stored at RT and 4°C for 3 months and reconstituted in 1x PBS. [Figure 8]Figure 1 shows a graph of the expression of SARS-CoV-2 spike protein-specific IgG in C57BL / 6 mice 42 days after IM administration of saRNA LNPs encoding SARS-CoV-2 spike protein at a dose of 1 μg / mouse containing PNI 516 in VB composition (N / P-8) after lyophilization, storage at three different temperatures (-20°C, 4°C, and RT) for one week, and reconstitution. [Figure 9] 1 is a graph showing human erythropoietin (hEPO) protein expression in HEK293 cells treated with a dose of 1 μg / mL of lyophilized LNPs stored for one week at three different temperatures (20° C., 4° C., and RT) and reconstituted in 1×PBS. [Figures 10A-10E] 10A and 10B show graphs showing EPO protein expression levels in HEK293 cells treated with lyophilized LNPs reconstituted in 1×PBS at a dose of 1 μg / mL, stored at RT for 1 week. These EPO mRNA-LNPs contain either PNI 516 (FIG. 10A), PNI 127 (FIG. 10B), dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) (FIG. 10C), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) (FIG. 10D), or butanoic acid, 4-(dimethylamino)-,9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester (BOCHD-C3-DMA) (FIG. 10E) in a VB composition N / P-8. [Figure 11A] 1 is a graph showing the encapsulation efficiency results of lyophilized EPO mRNA-LNPs formulated with PNI 516 and reconstituted in 1×PBS stored at RT for 1 week. [Figure 11B] 1 is a graph showing the encapsulation efficiency results of lyophilized EPO mRNA-LNPs formulated at PNI 127 and reconstituted in 1×PBS stored at RT for 1 week. [Figure 11C]1 is a graph showing the encapsulation efficiency results of lyophilized EPO mRNA-LNPs formulated with dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and reconstituted in 1×PBS stored at RT for 1 week. [Figure 11D] 1 is a graph showing the encapsulation efficiency results of lyophilized EPO mRNA-LNPs formulated with 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) and reconstituted in 1×PBS stored at RT for 1 week. [Figure 11E] 1 is a graph showing the encapsulation efficiency results of lyophilized EPO mRNA-LNPs formulated with butanoic acid, 4-(dimethylamino)-9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester (BOCHDC3-DMA) and reconstituted in 1x PBS, stored at RT for 1 week. [Figure 12A] 1 is a graph showing the size and polydispersity results of lyophilized EPO mRNA-LNPs formulated with PNI 516 and reconstituted in 1×PBS after 1 week of storage at RT. [Figure 12B] 1 is a graph showing the size and polydispersity results of lyophilized EPO mRNA-LNPs formulated at PNI 127 and reconstituted in 1×PBS after 1 week of storage at RT. [Figure 12C] 1 is a graph showing the size and polydispersity results of lyophilized EPO mRNA-LNPs formulated with dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and reconstituted in 1×PBS after 1 week of storage at RT. [Figure 12D] Graph showing size and polydispersity results of lyophilized EPO mRNA-LNPs formulated with 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) and reconstituted in 1x PBS after 1 week of storage at RT. [Figure 12E]Graph showing size and polydispersity results of lyophilized EPO mRNA-LNPs formulated with butanoic acid, 4-(dimethylamino)-9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester (BOCHD-C3-DMA) and reconstituted in 1x PBS after 1 week of storage at RT. [Figure 13A] 1 is a graph showing EPO protein expression levels in HEK293 cells treated with LNPs lyophilized and reconstituted in 1×PBS at a dose of 1 μg / mL after 1 week of storage at RT. PN1 516 and EPO mRNA-LNPs differed by different helper lipids (DSPC, DPPC, DOPE, or DOPC) in the VB composition (N / P-8) and were lyophilized using LB#107 buffer. [Figure 13B] 1 is a graph showing EPO protein expression levels in HEK293 cells treated with LNPs lyophilized and reconstituted in 1×PBS at a dose of 1 μg / mL after 1 week of storage at RT. LNPs were lyophilized with PNI 516 and the helper lipid DSPC for EPO mRNA-LNPs using LB# 110 and LB# 189 buffers; [Figure 13C] 1 shows graphs showing EPO protein expression levels in HEK293 cells treated with LNPs lyophilized and reconstituted in 1×PBS at a dose of 1 μg / mL after 1 week of storage at RT. LNPs of PNI 516 and EPO mRNA-LNPs with the helper lipid DPPC were lyophilized using LB#107 and LB#189 buffers. [Figure 14] 1 is a graph showing luciferase protein expression levels in HEK293 cells treated with LNPs containing PNI 516 or PNI 127 at a dose of 25 ng / well that were lyophilized and stored at RT for 1 week before being reconstituted in 1x PBS. [Figure 15]1 is a graph showing EPO expression levels in C57BL / 6 mice after intravenous administration of PN1 516 and mRNA LNPs encoding recombinant human EPO (VB composition, N / P-8) at a dose of 0.25 mg / kg, which were lyophilized prior to treatment, stored at three different temperatures (-20°C, 4°C, and RT) for 1 week, and then reconstituted in 1x PBS prior to administration. [Figure 16]

[0023] Figure 1 shows the encapsulation efficiency of lyophilized LNPs made from mRNA encoding PN1516 and EPO using three different lyophilization buffers after administration to mice. The lyophilized cakes were stored at three different temperatures (-20°C, 4°C, and RT) for one week and reconstituted in 1x PBS before treatment. [Figure 17] 1 shows the size and PDI results of lyophilized LNPs made from mRNA encoding PN1516 and EPO using three different lyophilization buffers after administration to mice. The lyophilized cakes were stored at three different temperatures (-20°C, 4°C, and RT) for one week and reconstituted in 1x PBS before treatment. [Figure 18] Western blot image of cell lysates from HEK-293 cells treated with lyophilized LNPs containing in vitro-expressed SARS-CoV-2 spike protein mRNA (0.25 μg / mL saRNA, 24 hours). The LNPs were lyophilized using a lyophilization buffer containing PNI 516 and LB# 3, 9, 31, 54, 98, 101, 112, 113, 119, 120, 134, 135, 136, 142, 143, 147, 148, 157, 161, 162, 163, 164, 166, 167, 168, 170, 180, 181, 182, 187, 188, and 208. The LNPs were stored at RT for 12 days and reconstituted in 1x PBS before treatment. [Figure 19] FIG. 1 shows a graphical representation of EPO protein levels in the serum of mice treated with lyophilized protected LNPs (LB#54, 108, 167, 218, 241, and 260) after 2 months of storage at 4° C. [Figure 20]1 is a graphical representation of SARS-CoV-2 spike protein-specific IgG levels in the serum of mice treated with dosing material containing lyophilization buffer Lead LB#354, 108, 164, and 167 after storing LNPs at 4°C for 1 month. DETAILED DESCRIPTION OF THE INVENTION

[0018] The lyophilized nucleic acid lipid nanoparticles (NALNPs) of the present invention comprise (a) lipid nanoparticles comprising a nucleic acid, and (b) a lyophilization buffer comprising a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent.

[0019] The lyophilized NALNP of the present invention comprises lipid nanoparticles containing nucleic acids. Lipid nanoparticles are a subgroup of lipid particles having an average diameter of about 15 to about 300 nm. In some embodiments, the average particle size is greater than 200 nm. In some embodiments, the lipid particles have a diameter of 300 nm or less, about 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the lipid particles have a diameter of about 50 to about 150 nm. Smaller particles generally exhibit increased circulatory life in vivo compared to larger particles. Smaller particles have an increased ability to reach tumor sites compared to larger nanoparticles. In one embodiment, the lipid particles have a diameter of about 15 to about 50 nm.

[0020] Generally, lipid nanoparticles are spherical aggregates of lipids, nucleic acids, sterols, and stabilizers. The positive and negative charges, ratios, and hydrophilic and hydrophobic properties influence the physical structure of lipid particles in terms of size and orientation of components. The structural organization of these lipids can result in an aqueous interior with a minimal bilayer, like liposomes, or a solid interior, like solid nucleic acid-lipid nanoparticles. The phospholipid monolayer or bilayer can exist in single or multiple forms. Lipid particles range in size from 1 to 1000 μm.

[0021] In some embodiments, the lipid nanoparticles comprise a lipid mixture solution and nucleic acid. In some embodiments, the lipid mixture solution comprises an ionizable lipid, a structured lipid, a sterol, and a stabilizer. "N / P" refers to the molar ratio of the amine groups of the ionizable lipid to the phosphate groups of the nucleic acid. In some embodiments, the N / P ratio is 4-12. In preferred embodiments, the N / P ratio is 6-10. For example, in preferred embodiments, the N / P ratio is 6, 8, or 10. The nucleic acid is associated with the lipid mixture composition to form LNPs with a pre-designed ratio of ionizable lipid amine (N) to nucleic acid phosphate (P), such as an N / P ratio of 4, 6, 8, 10, 12, or any other suitable N / P ratio. In some embodiments, the lipid mixture solution comprises a stabilizer or stabilizer. Any suitable stabilizer or stabilizer may be used in embodiments of the present invention. In some embodiments, the stabilizer is selected from polysorbate (Tween), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™ 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij™ S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), Myrj™ 52 (polyoxyethylene (40) stearate), PEG-DMG, PEG-DMG 2000, triolein, tridecyl-D-maltoside, Tween 20, polysorbate 80, lipid H, TPGS 1000, polyoxyethylene (4) lauryl ether, and DiD. In some embodiments, a combination of stabilizers is used, such as polysorbates and maltosides, alkyl polyglycosides (TBD), PEG-conjugated lipids, or other polymer-conjugated lipids. In some embodiments, the lipid mixture solution contains two or more stabilizers or stabilizers. For example, in some embodiments, the lipid mixture solution comprises one or more, two or more, three or more, or four or more stabilizers or stabilizing agents.

[0022] In some embodiments, the lipid mixture solution comprises ionizable lipid.Any suitable ionizable lipid can be used in the present invention.The ionizable lipid is a cationic lipid, or a lipid that becomes ionized (protonated) as the pH becomes lower than the pKa of the ionizable group of the lipid, but becomes more neutral at higher pH values.At pH values ​​below the pKa, the lipid can then associate with negatively charged nucleic acids (e.g., oligonucleotides).Examples of suitable ionizable lipids can be found in PCT Publication Nos. WO2020252589 and WO2021000041.

[0023] In some embodiments, the ionizable lipid is DODMA (1,2-dioleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino)), DLin-KC2-DMA (2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane), butanoic acid, BOCHD-C3-DMA (4-(dimethylamino)-,9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester), or C12-200. In some preferred embodiments, the ionizable lipid is (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butanoate (referred to as PNI 516) (WO 2020 / 252589) or (2R,3S,4R)-2-(((1,4-dimethylpiperidine-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl(9E,9′E,12E,12′E)-bis(octadeca-9,12-dienoate) (referred to as PNI 127) (WO 2021 / 000041).

[0024] In some embodiments, the lipid mixture solution includes a structured lipid. Structured lipids may also be known as helper lipids or neutral lipids. Any suitable structured lipid may be used in embodiments of the present invention. Suitable structured lipids support particle formation during production. A structured lipid refers to any one of a number of lipid species that exist in either anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative structured lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylglycerol, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0025] Exemplary structured lipids include zwitterionic lipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), palmitoyloleoylphosphatidylethanolamine (POPE), and dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In a preferred embodiment, the structured lipid is distearoylphosphatidylcholine (DSPC).

[0026] In another embodiment, the structured lipid is any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups linked to neutral lipids. Other suitable structured lipids include glycolipids (e.g., monosialoganglioside GM1).

[0027] In some embodiments, the lipid mixture solution includes a sterol. Any suitable sterol may be used. In some embodiments, the sterol is cholesterol, beta-sitosterol, 20-alpha-hydroxysterol, or phytosterol. In a preferred embodiment, the sterol is cholesterol.

[0028] The lipid mixture can include any suitable combination of ionizable lipids, structured lipids, sterols, and stabilizers. In some embodiments, the lipid mixture includes 47.5 mol% ionizable lipids, 12.5 mol% structured lipids, 38.5 mol% sterols, and 1.5 mol% stabilizers. In a preferred embodiment, the lipid mixture includes 47.5 mol% IL, 13.5 mol% DOPE, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. In another preferred embodiment, the lipid mixture includes 47.5 mol% IL, 122.5 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. In another embodiment, the lipid mixture includes 40 mol% ionizable lipids, 20 mol% structured lipids, 37.5 mol% sterols, and 2.5 mol% stabilizers. For example, in a preferred embodiment, the lipid mixture comprises 40 mol% ionizable lipid, 30 mol% DSPC, 37.5 mol% cholesterol, and 2.5 mol% BRIJ™ S10.

[0029] The lipid nanoparticles contain nucleic acids. Any suitable nucleic acid can be used in the lipid nanoparticles. Nucleic acids are substances intended to have a direct effect on the diagnosis, cure, alleviation, treatment, or prevention of disease, or to have a direct effect on restoring, correcting, or modifying physiological function, or to function as a research reagent. In some embodiments, the nucleic acid is siRNA, miRNA, self-amplifying RNA (SAM or saRNA), self-replicating DNA, LNA, DNA, replicon, mRNA, guide RNA, transposon, or single gene. In some embodiments, the nucleic acid is referred to as nucleic acid therapy or NAT.

[0030] The lyophilized NALNPs of the present invention comprise a lyophilization buffer (sometimes referred to as a lyo buffer) that includes a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent.

[0031] The lyophilization buffer of the present invention comprises a sugar. Any suitable sugar may be used. In some embodiments, the sugar is selected from sucrose, mannose, mannitol, sorbitol, raffinose, fructose, glucose, lactose, maltose, maltodextrin, trehalose, inulin, and dextran. In a preferred embodiment, the sugar is sucrose. In some embodiments, the lyophilization buffer comprises two or more types of sugar. In some embodiments, the lyophilization buffer comprises one or more, two or more, or three or more types of sugar.

[0032] The lyophilization buffer of the present invention includes a lyophilization reagent. In some embodiments, the lyophilization reagent is selected from polyvinyl alcohol, a sugar-mimetic oligomer / polymer, an amphiphilic thermoresponsive polymer, an ethylene glycol-mimetic polymer, or a hydrophilic monomer or polymer. In some embodiments, the sugar-mimetic oligomer / polymer is n-octanoylsucrose, cyclodextrin, beta-cyclodextrin, dextran, trehalose, sorbitol-core carboxy-terminated PEG, Betadex™ sulfobutyl ether sodium, or 2-hydroxypropyl-beta-cyclodextrin. In some embodiments, the amphiphilic thermoresponsive polymer is poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide). In some embodiments, the ethylene glycol-mimetic polymer is a 6-arm branched PEG, a 5-arm branched PEG, a 3-arm branched PEG, trimethylpropane ethoxylate, polyethylene glycol, Pluronic™ (F-127), an amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol). In some embodiments, the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), an amino acid, or L-arginine.

[0033] In a preferred embodiment, the lyophilization reagent is polyvinylpyrrolidone. In another preferred embodiment, the lyophilization reagent is 2-hydroxypropyl-beta-cyclodextrin. The lyophilization buffer of the present invention comprises a pharmaceutically acceptable diluent. Any suitable pharmaceutically acceptable diluent may be used. In some embodiments, the pharmaceutically acceptable diluent is selected from a solution of Tris, sodium acetate, dextrose, 5% dextrose, saline, PBS, lactated Ringer's solution, 5% human serum albumin, and water. In a preferred embodiment, the pharmaceutically acceptable diluent is Tris buffer. In some embodiments, the lyophilization buffer comprises two or more pharmaceutically acceptable diluents. For example, in some embodiments, the lyophilization buffer comprises one or more, two or more, or three or more types of pharmaceutically acceptable diluents. In a preferred embodiment, the lyophilization buffer comprises Tris buffer and PBS. In some embodiments, the salt concentration of the pharmaceutically acceptable diluent is 0 to 70 mg / mL. For example, in some embodiments, the salt concentration of the pharmaceutically acceptable diluent is 0 mg / mL, 5 mg / mL, 10 mg / mL 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, or 70 mg / mL, or between any two of the aforementioned values.

[0034] In some embodiments, the lyophilization buffer comprises a base composition buffer (also referred to as BC). In some embodiments, the base composition buffer comprises a sugar and a pharmaceutically acceptable diluent as described herein. For example, in a preferred embodiment, the base composition buffer comprises 10% (wt / vol) sucrose dissolved in 20 mM Tris buffer and 1×PBS (10 mg / mL). In some embodiments, the lyophilization buffer comprises a base composition buffer and a lyophilization reagent. In a preferred embodiment, the lyophilization buffer comprises the lyophilization reagent in an amount of 1 to 5% wt / vol. For example, in some embodiments, the lyophilization buffer comprises the lyophilization reagent in an amount of 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, or any value between any two of the aforementioned values.

[0035] In some embodiments, the lyophilization buffer has a weight / mass ratio of saccharide to lyophilization reagent of 1:1 to 1:20. For example, in some embodiments, the lyophilization buffer has a weight / mass ratio of saccharide to lyophilization reagent of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20, or a ratio between any two of the aforementioned values. In a preferred embodiment, the lyophilization buffer has a weight / mass ratio of saccharide to lyophilization reagent of 1:4 to 1:10. In another embodiment, the weight / mass ratio of saccharide to lyophilization reagent is 1:8.

[0036] In some embodiments, the lyophilization buffer has a weight / weight ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent of 1:1:40 to 1:10:80. For example, in some embodiments, the weight / weight ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent is 1:1:40, 1:2:40, 1:3:40, 1:4:40, 1:5:40, 1:6:40, 1:7:40, 1:8:40, 1:9:40, 1:10:40, or a range defined by any two of the foregoing values. In other embodiments, the weight / weight ratio of lyophilized reagent to sugar to pharmaceutically acceptable diluent is 1:1:80, 1:2:80, 1:3:80, 1:4:80, 1:5:80, 1:6:80, 1:7:80, 1:8:80, 1:9:80, 1:10:80, or a range defined by any two of the foregoing values. In other embodiments, the weight / weight ratio of lyophilized reagent to sugar to pharmaceutically acceptable diluent is 1:4:40, 1:4:45, 1:4:50, 1:4:55, 1:4:60, 1:4:65, 1:4:70, 1:4:75, 1:4:80, or a range defined by any two of the foregoing values.

[0037] In other embodiments, the weight / weight ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent is 1:8:40, 1:8:45, 1:8:50, 1:8:55, 1:8:60, 1:8:65, 1:8:70, 1:8:75, 1:8:80, or a range defined by any two of the foregoing values. In a preferred embodiment, the lyophilization buffer has a weight / weight ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent of 1:4:40 to 1:8:80.

[0038] In this application, the term "diluent" refers to either the liquid or the lyophilized form. Thus, in the lyophilized form, the "diluent" is the dehydrated residue of the diluent used in the lyophilization process.

[0039] In some embodiments, the NALNPs have a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0040] In some embodiments, the NALNP is in an anhydrous form. The NALNP is in an anhydrous form after being lyophilized as described herein. In some embodiments, the NALNP is in an anhydrous form consisting of lyophilized or lyocake. In some embodiments, the NALNP is in a reconstituted form. In the reconstituted form, the lyophilized NALNP has had a pharmaceutically acceptable diluent added to the lyophilized NALNP described herein.

[0041] The method of the present invention for preparing freeze-dried NALNPs includes the steps of: (a) mixing a nucleic acid with a lipid mixture solution containing ionizable lipids, structured lipids, sterols, and stabilizers to form lipid nanoparticles; (b) combining the lipid nanoparticles obtained in (a) with a freeze-drying buffer; and (c) freeze-drying the combination of lipid nanoparticles obtained in step (b) and the freeze-drying buffer to obtain anhydrous freeze-dried NALNPs. The method of the present invention for preparing freeze-dried NALNPs includes the step of mixing a nucleic acid with a lipid mixture solution containing ionizable lipids, structured lipids, sterols, and stabilizers to form lipid nanoparticles. The nucleic acid can be any suitable nucleic acid according to embodiments of the present invention. The lipid mixture solution containing ionizable lipids, structured lipids, sterols, and stabilizers can be any suitable lipid mixture according to embodiments of the present invention. The lipid nanoparticles can be any suitable lipid nanoparticles according to embodiments of the present invention.

[0042] Any suitable mixing method can be used to form lipid nanoparticles. Lipid nanoparticles according to embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, titration mixing, stirring to promote self-assembly, or passively mixing all components followed by self-assembly of the components into nanoparticles. Various methods have been developed to formulate lipid nanoparticles containing genetic drugs. Suitable methods are disclosed, for example, in U.S. Patent Nos. 5,753,613 and 6,734,171. These methods involve mixing preformed lipid particles with nucleic acid in the presence of ethanol, or mixing lipids dissolved in ethanol with an aqueous medium containing nucleic acid, yielding lipid nanoparticles with nucleic acid encapsulation efficiencies of 65-99%. Both of these methods utilize ionizable lipids to achieve nucleic acid encapsulation and the presence of stabilizers to inhibit aggregation and the formation of large structures.

[0043] Automated micromixing instruments, such as the NanoAssemblr® instrument (Precision NanoSystems, Vancouver, Canada), enable rapid and controlled production of nanomedicines (liposomes, lipid nanoparticles, and polymer nanoparticles). The NanoAssemblr® instrument achieves controlled molecular self-assembly of nanoparticles using microfluidic mixing cartridges that allow for millisecond mixing of nanoparticle components at nanoliter, microliter, or larger scales, either custom or parallel. Rapid mixing on a small scale allows for reproducible control over particle synthesis and quality that is not possible with larger instruments.

[0044] A preferred method incorporates equipment such as microfluidic mixing devices such as NanoAssemblr® Spark™, ​​Ignite™, Benchtop™, and NanoAssemblr® Blaze™ to achieve encapsulation of nearly 100% of the nucleic acid used in the formation process within the particles in a single step. In one embodiment, the lipid particles are prepared by a process in which about 90 to about 100% of the nucleic acid used in the formation process is encapsulated within the particles.

[0045] U.S. Patent Nos. 9,758,795 and 9,943,846 describe methods using small-volume mixing technology and novel formulations derived therefrom. U.S. Patent No. 10,159,652 describes more advanced methods using small-volume mixing technology and products for formulating different materials. U.S. Patent No. 9,943,846 discloses a microfluidic mixer with channels and wells distinct from the elements to be mixed, and International Publication No. 2017117647 discloses a microfluidic mixer with a disposable, sterile channel. U.S. Patent No. 10,076,730 discloses a branched, annular micromixing geometry and its application to micromixing. International Publication No. 2018006166 discloses a programmable, automated micromixer and a mixing chip therefor. Mixing cartridges with microchannels and mixing geometries for mixing devices are commercially available, for example, from Precision NanoSystems.

[0046] In an embodiment of the invention, a biological microfluidic mixing device is used to prepare lipid particles according to an embodiment of the invention, the device comprising first and second reagent streams that are fed into a microfluidic mixer, and lipid particles that are collected from an outlet or emerge into a sterile environment.

[0047] The first stream contains a therapeutic agent in a first solvent. Suitable first solvents include those in which the therapeutic agent is soluble and which are miscible with the second solvent. Suitable first solvents include aqueous buffers. Exemplary first solvents include citrate and acetate buffers or other low pH buffers.

[0048] The second stream contains the lipid mixture material in a second solvent. Suitable second solvents include those in which the ionizable lipids according to embodiments of the present invention are soluble and miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Exemplary second solvents include aqueous 90% ethanol or absolute ethanol.

[0049] In one embodiment of the present invention, a suitable device includes one or more microchannels (i.e., the largest dimension of the channel is less than 1 millimeter). In one example, the microchannel has a diameter of about 20 to about 300 μm. In one example, at least one region of the microchannel has one or more surfaces with a primary flow direction and at least one groove or protrusion defined therein, the groove or protrusion being oriented at an angle to the primary flow direction (e.g., a twisted herringbone mixer) as described in U.S. Pat. No. 9,943,846, or a branched annular flow as described in U.S. Pat. No. 10,076,730. To achieve maximum mixing rates, it is advantageous to avoid excessive fluid resistance before the mixing region. Thus, one example of a device includes non-microfluidic channels with dimensions greater than 1000 μm to deliver liquids to a single mixing channel.

[0050] Less automated mixing methods and equipment, such as those disclosed in Zhang, Sh et al., Chemical Eng. J. 144(2): 324-328 (2008) and U.S. Patent Application No. 20040262223, and Jeffs, LB et al., Pharm. Resch., 22(3): 362-372 (2005), are also useful in creating the lipid particle compositions of the present invention.

[0051] The method of the present invention for preparing lyophilized NALNPs includes combining lipid nanoparticles with a lyophilization buffer. Any suitable lyophilization buffer according to embodiments of the present invention may be used. Any suitable method for combining lipid nanoparticles with a lyophilization buffer may be used. For example, in some embodiments, the lyophilization buffer and lipid nanoparticles are mixed. In a preferred embodiment, the lyophilization buffer and lipid nanoparticles are mixed using a pipette.

[0052] The method of the present invention for preparing lyophilized NALNP includes freeze-drying a combination of lipid nanoparticles and a freeze-drying buffer to obtain anhydrous, freeze-dried NALNP. In some embodiments, the freeze-drying step includes (a) freezing the combination of lipid nanoparticles and a freeze-drying buffer at −40 to −90°C for 60 to 400 minutes, (b) drying the combination of lipid nanoparticles and a freeze-drying buffer at −20 to −40°C and 30 to 100 mTorr for 700 to 980 minutes, and (c) drying the combination of lipid nanoparticles and a freeze-drying buffer at 4 to 10°C and 30 to 100 mTorr for 250 to 500 minutes.

[0053] Lyophilization of the combination of nanoparticles and lyophilization buffer can include freezing the combination of lipid nanoparticles and lyophilization buffer. Any suitable method for freezing the lipid nanoparticles and lyophilization buffer can be used. For example, the combination of lipid nanoparticles and lyophilization buffer can be placed directly in a subzero freezer, frozen in liquid nitrogen (e.g., by immersion for an appropriate period of time, such as about 30 seconds), or placed in a controlled freezing container and then placed in a freezer. The combination of nanoparticles and lyophilization buffer can be frozen at any suitable temperature. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is frozen at -40 to -90°C. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is frozen at -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, -85°C, -90°C, or within a range defined by any two of the aforementioned values.

[0054] In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is frozen at -60°C or -80°C. The combination of lipid nanoparticles and lyophilization buffer can be frozen for any suitable period of time. In some embodiments, the combination of nanoparticles and lyophilization buffer is frozen for 60 to 400 minutes. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is frozen for 60 minutes, 75 minutes, 100 minutes, 125 minutes, 150 minutes, 175 minutes, 200 minutes, 225 minutes, 250 minutes, 275 minutes, 300 minutes, 325 minutes, 350 minutes, 375 minutes, 400 minutes, or a period within a range defined by any two of the aforementioned values.

[0055] Lyophilization of the combination of nanoparticles and lyophilization buffer can include drying or dehydrating the combination of lipid nanoparticles and lyophilization buffer. In some embodiments, the combination of nanoparticles and lyophilization buffer is dried once. In some embodiments, the combination of nanoparticles and lyophilization buffer is dried two or more times. For example, the combination of nanoparticles and lyophilization buffer can be dried once, twice, three times, four times, or five times. Any suitable method for drying the combination of lipid nanoparticles and lyophilization buffer can be used.

[0056] In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is first dried at -20 to -40°C and 30 to 100 mTorr for 700 to 980 minutes. The combination of lipid nanoparticles and lyophilization buffer can be dried at any suitable temperature. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer can be dried at a temperature of -20 to -40°C. For example, the combination of lipid nanoparticles and lyophilization buffer can be dried at a temperature of -20°C, -22°C, -24°C, -26°C, -28°C, -30°C, -32°C, 34°C, -36°C, -38°C, -40°C, or within a range of any two of the aforementioned values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried at a temperature of -40°C. The combination of lipid nanoparticles and lyophilization buffer can be dried at any suitable pressure. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 30 to 100 mTorr. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 30 mTorr, 35 mTorr, 40 mTorr, 45 mTorr, 50 mTorr, 55 mTorr, 60 mTorr, 65 mTorr, 70 mTorr, 75 mTorr, 80 mTorr, 85 mTorr, 90 mTorr, 95 mTorr, 100 mTorr, or a range between any two of the aforementioned values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 60 mTorr. The combination of lipid nanoparticles and lyophilization buffer can be dried for any suitable period of time. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried for 700 to 980 minutes. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried for 700 minutes, 720 minutes, 740 minutes, 760 minutes, 780 minutes, 800 minutes, 820 minutes, 840 minutes, 860 minutes, 880 minutes, 900 minutes, 920 minutes, 940 minutes, 960 minutes, 980 minutes, or within a range of any two of the foregoing values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried for 840 minutes.

[0057] In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried a second time. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried a second time at 4-10°C and 30-100 mTorr for 250-500 minutes. The combination of lipid nanoparticles and lyophilization buffer can be dried a second time at any suitable temperature. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer can be dried at a temperature of 4-10°C. For example, the combination of lipid nanoparticles and lyophilization buffer can be dried at a temperature of 4°C, 4.5°C, 5°C, 5.5°C, 6°C, 6.5°C, 7°C, 7.5°C, 8°C, 8.5°C, 9°C, 9.5°C, 10°C, or a temperature within a range of any two of the aforementioned values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried at a temperature of 10°C. The combination of lipid nanoparticles and lyophilization buffer can be dried at any suitable pressure. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 30 to 100 mTorr. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 30 mTorr, 35 mTorr, 40 mTorr, 45 mTorr, 50 mTorr, 55 mTorr, 60 mTorr, 65 mTorr, 70 mTorr, 75 mTorr, 80 mTorr, 85 mTorr, 90 mTorr, 95 mTorr, 100 mTorr, or a range between any two of the aforementioned values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried at a pressure of 60 mTorr. The combination of lipid nanoparticles and lyophilization buffer can be dried for any suitable period of time. In some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried for 250 to 500 minutes. For example, in some embodiments, the combination of lipid nanoparticles and lyophilization buffer is dried for 250 minutes, 260 minutes, 280 minutes, 300 minutes, 320 minutes, 340 minutes, 360 minutes, 380 minutes, 400 minutes, 410 minutes, 440 minutes, 460 minutes, 480 minutes, 500 minutes, or within any two of the foregoing values. In a preferred embodiment, the combination of lipid nanoparticles and lyophilization buffer is dried for 320 minutes.

[0058] In some embodiments, the method further comprises reconstituting the anhydrous lyophilized NALNP. Any suitable method can be used to reconstitute the anhydrous lyophilized NALNP. In some embodiments, the anhydrous lyophilized NALNP is combined with the solution until the resulting solution is visibly homogeneous. In some embodiments, the anhydrous lyophilized NALNP is combined with the solution for at least 30 minutes. For example, in some embodiments, the anhydrous lyophilized NALNP is combined with the solution for at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 110 minutes, at least 120 minutes, or within a range of any two of the aforementioned values. In some embodiments, the anhydrous lyophilized NALNP is combined with the solution at a temperature between 0°C and 10°C. For example, in some embodiments, the anhydrous, lyophilized NALNP is combined with the solution at a temperature of 0° C., 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C., or within a range of any two of the foregoing values. In a preferred embodiment, the anhydrous, lyophilized NALNP is combined as a solution at a temperature of 4° C. In some embodiments, the solution is a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is Tris, sodium acetate, dextrose, saline, or water.

[0059] Embodiment 1. (a) lipid nanoparticles containing nucleic acids, and (b) a lyophilization buffer solution comprising a sugar and a lyophilization reagent selected from a sugar-mimetic oligomer / polymer, an amphiphilic thermoresponsive polymer, an ethylene glycol-mimetic polymer, a hydrophilic monomer, and a hydrophilic polymer; Lyophilized nucleic acid lipid nanoparticles (NALNPs) comprising:

[0060] 2. The lyophilization buffer of embodiment 1, wherein the sugar-mimetic oligomer / polymer is n-octanoylsucrose, cyclodextrin, beta-cyclodextrin polymer, dextran, trehalose, sorbitol-core carboxy-terminated PEG, Betadex™ sulfobutyl ether sodium, or 2-hydroxypropyl-beta-cyclodextrin.

[0061] 3. The lyophilization buffer of embodiment 1, wherein the amphiphilic thermoresponsive polymer is poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide).

[0062] 4. The lyophilization buffer of embodiment 1, wherein the ethylene glycol-mimetic polymer is a 6-arm branched PEG, a 5-arm branched PEG, a 3-arm branched PEG, trimethylpropane ethoxylate, polyethylene glycol, poloxamer 407, an amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol).

[0063] 5. The lyophilization buffer of embodiment 1, wherein the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), an amino acid, or L-arginine.

[0064] 6. The NALNP of embodiment 2, wherein the mass / mass ratio of lyophilization reagent to sugar is 1:8.

[0065] 7. The NALNP of embodiment 2, wherein the mass / mass ratio of lyophilization reagent to sugar is 1:5.

[0066] 8. The NALNP of embodiment 1, further comprising a pharmaceutically acceptable diluent selected from Tris, sodium acetate, sodium citrate, dextrose, and saline solution.

[0067] 9. The NALNP of embodiments 1-8, wherein the NALNP has a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0068] 10. The NALNP of embodiment 1, wherein the sugar is sucrose.

[0069] 11. The NALNP of any one of embodiments 1-8, wherein the NALNP is in anhydrous form.

[0070] 12. (a) mixing a nucleic acid with a lipid mixture solution containing an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles; (b) combining the lipid nanoparticles obtained in (a) with a lyophilization buffer containing a sugar and a lyophilization reagent selected from the group consisting of polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-beta-cyclodextrin, N,N-dimethylacrylamide, poly(N,N-diethylacrylamide), poly(2-ethyl-2-oxazoline), glycerol ethoxylate, amine-terminated 4-arm PEG, 6-arm branched PEG, 5-arm branched PEG, and sorbitol-core carboxy-terminated PEG, and a pharmaceutically acceptable diluent; (c) lyophilizing the combination of lipid nanoparticles and lyophilization buffer obtained in step (b) to obtain anhydrous lyophilized NALNPs. A method for preparing freeze-dried NALNP, comprising:

[0071] 13. The step of lyophilizing the combination of nanoparticles and a lyophilization buffer comprises: (a) freezing a combination of lipid nanoparticles and a lyophilization buffer at −40 to −90° C. for 60 to 400 minutes; (b) drying the combination of lipid nanoparticles and lyophilization buffer at −20 to −40° C. and 30 to 100 mTorr for 700 to 980 minutes; (c) drying the combination of lipid nanoparticles and lyophilization buffer at 4-10°C and 30-100 mTorr for 250-500 minutes; 13. The method of embodiment 12, comprising:

[0072] 14. The method of any one of embodiments 12, wherein the lyophilization buffer comprises a pharmaceutically acceptable diluent.

[0073] 15. The method of embodiment 14, wherein the pharmaceutically acceptable diluent is Tris, sodium acetate, sodium citrate, dextrose, saline, or water.

[0074] 16. The method of any of embodiments 10-15, wherein the lyophilization buffer has a mass / mass ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent of 1:4:40 to 1:8:80.

[0075] 17. The method of any of embodiments 10-15, wherein the NALNP has a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0076] 18. The method of any of embodiments 10-15, wherein the sugar is sucrose.

[0077] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

[0078] All solvents and reagents were commercial products and were used as received unless otherwise stated. Temperatures are given in degrees Celsius. The following abbreviations are used in connection with the examples: hEPO-mRNA: human erythropoietin protein mRNA FLuc-mRNA: Firefly luciferase protein mRNA eGFP-mRNA: enhanced green fluorescent protein mRNA LB: Lyophilization buffer (lyo buffer) saRNA: self-amplifying mRNA eGFP: basic (constitutively fluorescent) green fluorescent protein from Equalea victoria hEPO: human erythropoietin h: time HPLC: High-performance liquid chromatography MFI: Median Fluorescence Intensity min:minutes mL: milliliter mmol: millimolar μL: microliter PBS: phosphate buffered saline wt: mass °C or degrees C: Celsius IL: ionizable lipid MC3:DLin-MC3-DMA Tween 80: Polysorbate 80 Brij™ L4: Polyoxyethylene (4) Lauryl Ether Brij™ S10: Polyoxyethylene (10) stearyl ether Brij™ S20: Polyoxyethylene (20) stearyl ether Brij™ S35: Polyoxyethylene (23) Lauryl Ether TPGS 1000: D-α-Tocopherol polyethylene glycol 1000 succinate VA composition (VA): lipid mixture containing 47.5 mol% IL, 12.5 mol% DOPE, 38.5 mol% cholesterol and 1.5 mol% PEG-DMG. VB composition (VB): A lipid mixture containing 47.5 mol% IL, 12.5 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG-DMG. CT10 composition: lipid mixture containing 40 mol% ionizable lipid / 20 mol% DSPC / 37.5 mol% cholesterol / 2.5 mol% Brij™ S10. PNI 516: (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadecan-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butanoate (WO 2020 / 252589). PNI 127: (2R,3S,4R)-2-(((1,4-dimethylpiperidine-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl(9E,9'E,12E,12'E)-bis(octadeca-9,12-dienoate) (WO 2021 / 000041). DLin-MC3-DMA or MC3: Dilinoleylmethyl-4-dimethylaminobutyrate. DLin-KC2-DMA or KC2: 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolambutanoic acid. BOCHD-C3-DMA: 4-(dimethylamino)-,9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester. V101: Cloning vector template for synthesizing self-amplifying replicon DNA or RNA encoding a gene of interest (GOI): parental VEEV TC83 replicon with a subgenomic promoter containing multiple cloning sites for insertion of any GOI (U.S. Patent No. 7,425,337 to Geall et al.). A3, A5: saRNA encoding the SARS-CoV-2 spike protein using the V101 vector (U.S. Patent No. 7,425,337). Base composition buffer (BC): 10% (wt / vol) sucrose dissolved in 20 mM Tris buffer and 1×PBS (10 mg / mL). Lyo Reagent or Lyophilized Reagent: The components are prepared in lyophilized buffer (LB). Lyophilization buffer or lyo buffer: The combination of lyo reagents [1-5% w / v, as listed in Table 1] in base composition (BC) buffer is defined as lyo buffer or lyophilization buffer (LB).

[0079] [Table 1A]

[0080] [Table 1B] [Example]

[0081] This example demonstrates the nucleic acid therapy (NAT) preparations used in the examples of this application. Messenger RNA, saRNA, or DNA plasmid nucleic acid therapy (NAT) was diluted to the required concentration using sodium acetate buffer as described below. RNA / pDNA was dissolved in 100 mM sodium acetate buffer to reach the desired concentration of approximately 168 μg / mL. The concentration of NAT was determined by Nanodrop (Thermo Scientific™). This information was used to determine the desired concentration of NAT for mixing with Lipid Mix in ethanol in the NanoAssemblr® Ignite™ instrument as described in Example 2. [Example]

[0082] This example demonstrates the preparation of lipid nanoparticles (LNPs) by microfluidic mixing, as used in the examples of this application. NATs were prepared as shown in Example 1. The components of the lipid mixture included ionizable lipids, structural or helper lipids, sterols, and stabilizers in different molar ratios. Stabilizers refer to any agent, including PEG-DMG or any agent under its category defined in the above description, for stabilizing LNP formation. Lipid mixture compositions were prepared in ethanol by combining predetermined amounts of lipids from individual lipid stocks in ethanol (12.5, 25, or 37.5 mM, as appropriate; mixtures of ionizable lipids, structural lipids, sterols, and stabilizers listed in Table 2). LNPs were then prepared by flowing the lipid mixture composition and NATs through a NanoAssemblr® Ignite™ microfluidic mixer.

[0083] Mixing of nucleic acid therapeutics (NATs) and lipids occurred as follows: Ionizable lipids, helper lipids, sterols, and stabilizers were mixed in 100% ethanol at a molar ratio of 47.5:12.5:38.5:1.5 (VA or VB) or 40:20:37.5:2.5 (CT10). The aqueous phase was prepared by diluting the nucleic acid therapeutics (NATs), such as mRNA / saRNA / pDNA solutions, in 100 mM sodium acetate buffer (pH 4). Solutions were combined using a NanoAssemblr® Ignite™ with an Ignite™ NxGen™ (DVBM) cartridge (Precision Nanosystems) at a flow ratio of 1:3 (organic phase:aqueous phase), an N / P ratio of 8 or 10, and a total flow rate of 12 mL / min, unless otherwise noted. The resulting LNPs were diluted 25-40 times in 1x PBS (pH 7.4), and the mixture was subjected to downstream processing, which included ethanol removal by dialysis in PBS (pH 7), using Amicon™ centrifugal filtration membranes (Millipore, USA) at 2500 RPM, or using a tangential flow filtration system. The particles were concentrated to the required target dose.

[0084] [Table 2A]

[0085] [Table 2B]

[0086] [Table 2C] [Example]

[0087] This example describes the methods used in the following examples to measure the size, polydispersity index (PDI) and encapsulation efficiency (EE) of LNPs.

[0088] The size and PDI of LNPs were measured by dynamic light scattering (DLS) using a ZetaSizer™ Nano ZS™ (Malvern Instruments). A 633 nm He / Ne laser was used as the light source. Data were measured from scattered intensity data run in backscatter detection mode (measurement angle = 173°). 0.5–2 μL of sample was placed in a cuvette and diluted with PBS (0.3 mL). Measurements were the average of 10 runs, each with two cycles per sample. The Z-average size was reported as particle size, and the harmonic intensity was defined as the average particle size. The EE of LNPs was measured using Quant-iT™ RiboGreen® RNA Reagent. These LNP characteristics, as well as nucleic acid EE results for LNPs in various lyophilization buffers (LB), are described in the following examples. [Example]

[0089] This example describes the lyophilization process and the methods used to evaluate the lyophilization buffers in the following examples.

[0090] Lyophilization buffer (lyo buffer) was prepared in the base composition (BC, 10% sucrose in 20 mM Tris and 1x PBS). The mass / volume percentage of each lyo reagent in the base composition is listed in Table 3. Once the LNPs reached the desired target concentrations (40, 80, and 120 g / mL) after preparation as described in Example 2, they were mixed with lyo buffer in ratios of 1:1, 1:2, and 1:4 (volume:volume). Final solution volumes of 200–300 μL were transferred into UPLC 2 mL glass vials or Afton Ready-To-Fill® sterile vials (2 mL). Once the required concentration was achieved, the LNPs mixed with lyo buffer were filter-sterilized using a 0.2 μm filter under aseptic conditions.

[0091] The LNPs mixed with lyo buffer were frozen at -60°C for 3 hours using a freeze-dryer (SP Scientific, model number ADP-S2XL-E0A-X; serial number 326328) and then lyophilized by first drying at -40°C / 0 min / 840 min / 60 mTorr, followed by a second drying at 10°C / 0 min / 320 min / 60 mTorr. A representative schematic of the lyophilization process workflow is shown in Figure 1, which details the different drying cycles. After secondary drying was completed, the resulting lyo cakes or lyo cakes were stored at different temperatures (RT, 4°C, or -20°C) for 24 hours, 1 week, 3 months, or 6 months (examples below). After the desired storage time, unless otherwise noted, the lyo cakes were reconstituted in 1x PBS at the target NAT concentration / volume (1:1 vol / vol) before treating any cells or animals as described in the following examples. After reconstitution, the final encapsulation efficiency was measured by Quant-iT™ RiboGreen® RNA reagent and kit (Invitrogen) according to the manufacturer's instructions, and the size of the LNPs was measured by dynamic light scattering (DLS) using a ZetaSizer™ Nano ZS™ (Malvern Instruments).

[0092] To evaluate the efficacy of lyo buffer, LNP formulations were prepared using different mRNAs (eGFP mRNA, hEPO mRNA, or FLuc mRNA), saRNA (A3 or A5), or pDNA in either 47.5 mol% IL / 12.5 mol% DOPE / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG (VA), 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG (VB), or 40 mol% ionizable lipid / 20 mol% DSPC / 37.5 mol% cholesterol / 2.5 mol% BRIJ™ S10 (CT10) compositions, as described in the following examples. The efficacy of lyo buffer was evaluated using LNPs containing various ionizable lipids and helper lipids encoding either hEPO mRNA or FLuc mRNA. The lyophilized compositions were compared to the base composition ("BC", in solution form, or 10% sucrose in 20 mM Tris and 1x PBS when reconstituted) either as lyophilized LNP cakes or frozen suspensions (BC, -80°C) where possible, or to fresh LNP (not lyophilized) in PBS.

[0093] [Table 3A]

[0094] [Table 3B]

[0095] [Table 3C] [Example]

[0096] To find a successful lyophilization protocol for mRNA vaccines, various lyobuffer compositions were evaluated for delivery of saRNA encoding vaccine antigens, using saRNA encoding the full-length spike protein. Physicochemical properties, such as size, polydispersity index (PDI), and encapsulation efficiency (EE), were measured for lyophilized LNPs (post-lyo) and compared with BC controls (BC plus lyophilized LNPs) and fresh LNPs (not lyophilized).

[0097] LNPs containing PN1516 as an ionizable lipid encapsulating saRNA encoding the spike antigen (SARS-CoV-2 spike protein, either A3b, A3p, or A5) were manufactured at target concentrations of 40, 80, and / or 120 μg / mL and diluted in 200 μL solution volumes in 2 mL glass UPLC vials at 1:1, 1:2, and 1:4 LNP to lyo buffer (volume:volume). The LNP formulation and lyo buffer compositions are described below. LNPs were lyophilized using different lyo buffers added to a base composition of 10% sucrose and 20 mM Tris. Dehydration was performed by freezing at -60°C for 3 hours, followed by dehydration at -40°C / 0 min / 840 min / 60 mTorr, followed by a second dehydration at 10°C / 0 min / 320 min / 60 mTorr. The lyophilized cakes were stored at RT for 24 hours. Additional tests were performed on LNPs containing A3 saRNA on lyocakes stored at -20°C, 4°C, and RT for 1 week. Reconstitution was performed in 200 L of PBS.

[0098] After reconstitution in 1x PBS, size, PDI, and encapsulation efficiency were measured. LB#107, 163, 186, 189, and 192 performed well in protecting the size and EE of LNPs containing PN1516 encapsulated with A3 saRNA within a therapeutically relevant range (Table 4). When the lyo cakes were stored at RT for 24 hours, LB#107, 110, 164, 168, 178, 179, 181, 182, 186, and 189 performed well in protecting the size and EE of LNPs containing PN1516 and A5 saRNA (Table 5). Figures 2 and 3 show the size, PDI, and EE of A3 saRNA LNPs using LB#107 and LB#110. Both lyo buffers (LB#107 and #110) protected the size and EE of LNPs within a therapeutically relevant range. Regardless of the payload tested, LB#110 was found to have the least size change. LB#189 possessed the lowest PDI, as shown in Table 4.

[0099] Some of the LBs in Table 1, such as sucrose alone, were not pursued further due to limitations in the initial results. For example, LB#3 (sucrose, 25%) was highly viscous. Furthermore, based on the data achieved, increasing the sucrose % from 10% to 25% in the absence of additional lyophilization reagents did not result in any improvement in CQA and potency. 10% sucrose in Tris 20 mM buffer was established as the base composition (BC) for adding the lyophilized drug and was used as the experimental control.

[0100] [Table 4]

[0101] [Table 5] [Example]

[0102] This example demonstrates the efficacy of lyophilized saRNA LNPs using Western blot analysis. LNPs containing PN1516, encoding the SARS-CoV-2 full-length spike protein, were formulated in a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG composition according to the standard formulation procedure described in the previous example. To maintain the in vitro efficacy of the saRNA LNPs, various lyo-buffer compositions were tested. The lyophilized cakes were then reconstituted in 1x PBS, sterilized, analyzed (size and EE), and used for in vitro cell therapy. Lyophilized LNPs in BC composition (BC) and PBS treatment (no LNPs) were used as controls.

[0103] Western blot: HEK-293 cells were seeded (0.3 × 10 ) in 6-well plates for 2 days. 6 The cells were then treated with 1 μg / mL of lyophilized LNPs (1000 cells / well) for 1 day. Cell lysates were prepared using IP lysis buffer. Protein quantification was performed using a BCA kit according to the supplier's protocol (Pierce™ BCA Protein Assay Kit). Protein samples were denatured using loading dye and 2-mercaptoethanol. SDS-PAGE was performed in a Mini Gel Tank (175 V, 500 mA, 90 min). The gel was transferred to a nitrocellulose membrane (Thermo Fisher) using an iBlot™ 2 device (Thermo Fisher). After blocking, the membrane was stained with primary antibody for 2 hours at RT on a shaker, followed by secondary antibody staining. The blot was developed using a chemiluminescent substrate. The blot was imaged using an iBright instrument (Thermo Fisher). Spike protein expression was qualitatively determined, and the performance of the lyophilized LNPs was assessed compared to the control.

[0104] Figure 4 shows Western blots of cellular proteins from treated cells. Lyophilized LNPs using LB#107, 110, 163, 164, 180, 186, and 189 performed equally well or better than BC lyophilized cakes. In this case, the lyocakes were stored at RT for 24 hours. In a separate experiment, lyophilized A3 saRNA LNP cakes were stored at three different temperatures (-20°C, 4°C, and RT) for one week. As shown in Figures 5 and 6, LB#163 demonstrated superior spike protein expression at all three temperatures. Figures 5 and 6 also suggest that LB#107, 186, and 192 similarly maintained saRNA activity at -20°C and 4°C. To assess the long-term efficacy of LB#163 in vitro, A3 saRNA LNP lyocakes containing LB#163 were stored at 4°C and RT for three months. LB#163 protected saRNA activity compared to BC at both temperatures, especially at 4° C. The results are shown in FIG. [Example]

[0105] This example demonstrates the ability of lyophilization buffer to maintain the activity of saRNA LNP vaccines in mice. Mice were administered (IM) lyophilized LNPs containing the ionized lipid PN1516 and encoding the SARS-CoV-2 full-length spike protein (A5 saRNA) reconstituted in 1x PBS using a VB composition. The lyophilized vaccine cakes were stored at -20°C, 4°C, and RT for 7 days. LNPs cryopreserved with PBS and BC were used as controls. Blood was collected from the tail vein after 42 days, and serum was prepared according to standard procedures. Six weeks later, serum analysis of SARS-CoV-2 spike protein-specific IgG was performed using established ELISA procedures.

[0106] Nunc Maxisorp™ ELISA plates were coated overnight with 0.5 μg / mL SARS-CoV-2 (2019-nCoV) Spike S1+S2 ECD-His Recombinant Protein (Cat. No. 40589-V08B1, Sino Biologicals, Beijing, China) in PBS. Mouse serum was diluted (1:40,000) using 1× ELISA Assay Diluent B (5×) (Cat. No. 421205, BioLegend, San Diego, USA). Standards were prepared (10–0.16 ng / mL) using SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody, Mouse MAb (Cat. No. 40591-MM43, Sino Biologicals). Detection was performed using HRP goat anti-mouse IgG (minimal x-reactivity) antibody (catalog no. 405306, BioLegend). Color development was performed using Western TMB substrate, stopped using HCl, and measurements were recorded at 450 nm. Figure 8 shows the measurement of SARS-CoV-spike protein-specific IgG. LB#163 performed equally well compared to the cryo-stored LNP control at all three different storage temperatures (-20°C, 4°C, and RT). [Example]

[0107] This example demonstrates the ability of the lyophilization buffer of the present invention to protect mRNA LNPs encoding human EPO protein (hEPO).

[0108] Culture conditions for HEK 293 cells for EPO expression: HEK 293 cells were cultured at 0.3 × 10 in 2 mL of complete DMEM (Gibco). 6Cells were seeded at 1 cell / well in 6-well plates and grown at 37°C with 5% CO2 for 48 hours. Lyophilized hEPO mRNA-LNPs were stored at the desired temperature for 24 hours, reconstituted in 1x PBS, and added to cells at 1 μg / mL mRNA along with controls. After 48 hours of incubation, the cell suspension was collected and centrifuged at 1200 g for 5 minutes. The supernatant was analyzed to measure hEPO protein concentration using a Simple Plex™ Human Erythropoietin Cartridge on an ELLA™ instrument (Biotechne, Protein Simple™).

[0109] Lyophilization: LNPs encoding hEPO mRNA were formulated as described in the examples above. To maintain the in vitro potency of mRNA LNPs, four lead lyobuffer compositions formulated in the VB composition (47.5 mol% PNI 516 / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG) were tested. LNPs lyophilized in BC or PBS treatment (no LNP) were used as controls. Lyophilized cakes were stored at different temperatures (RT and 4°C) for 24 hours, reconstituted in 1x PBS, sterilized, analyzed (size and EE), and used for cell treatment. The results are shown in Figure 9. Fresh LNPs had better activity than LNPs lyophilized in BC buffer, indicating that a basic composition containing 20 mM Tris and 10% sucrose cannot protect mRNA when LNPs are lyophilized. Lyophilized and reconstituted LNPs in lyo buffer (LB #163, #189, #110, and #107) retained mRNA activity and performed better than the BC buffer composition at both temperatures (4°C and RT). The size and encapsulation efficiency (%) of the corresponding lyophilized LNPs were measured and are shown in Table 6.

[0110] [Table 6] [Example]

[0111] This example demonstrates the ability of the lyophilization buffer of the present invention to protect mRNA LNPs encoding human EPO protein (hEPO).

[0112] In another study conducted with hEPO mRNA, the encapsulation efficiency (%) and size data of reconstituted (1x PBS) lyocakes of PN1516 and hEPO LNPs were measured after storing the lyocakes at the designated temperatures (-20°C, 4°C, or RT) for 7 days. LNP formation and lyophilization were as described in Example 8. The results are provided in Table 7. Fresh LNPs in BC and lyophilized LNPs at the respective temperatures (-20°C, 4°C, or RT) are shown as positive controls. LBs 107, 110, 163, 186, and 189 maintained LNP size and EE in the therapeutic range (Table 7).

[0113] [Table 7] [Example]

[0114] This example demonstrates the effectiveness of lyo buffers using various lipid mixture compositions by varying either the ionizable lipid (IL) or the helper lipid (HL). LNPs were prepared using different ionizable lipids and / or different helper lipids to evaluate the effectiveness of the lyophilization buffer in protecting mRNA. Lyophilized LNPs were stored under appropriate test conditions and / or times, and RNA integrity was tested using an in vitro efficacy assay in HEK-293 cells.

[0115] Lyophilization. 100 μL of LNP encapsulating hEPO mRNA at 80 μg / mL was mixed with 100 μL of buffer and lyophilized. After lyophilization, the cake was stored at RT for 7 days. The cake was reconstituted in 1×PBS, and the size and encapsulation efficiency (%) were determined. LNP stored at −80°C using BC was used as a positive control.

[0116] hEPO mRNA-LNPs were encapsulated using various ionizable lipids, PNI 516, PNI 127, MC3, KC2, and BOCHD-C3-DMA, in a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG composition, as well as controls such as fresh LNPs (without lyophilization) and BC lyophilized LNPs. The lyo cakes were stored at room temperature for 7 days and reconstituted in 1x PBS. The results are shown in Figure 10, with lyo buffers LB#189, 110, and 107 preserving EPO expression better than BC and fresh LNPs. The corresponding sizes and encapsulation efficiencies (%) were measured and are shown in Figures 11 and 12.

[0117] hEPO mRNA-LNPs were prepared with various helper lipids, DSPC, DOPE, DPPC, or DOPC, at a molar ratio of PNI516:cholesterol:helper lipid:PEG-DMG (47.5:38.5:12.5:1.5), and lyophilized. Lyophilized cakes were stored at room temperature for 7 days, reconstituted with 1x PBS, and analyzed for size and encapsulation efficiency (%) (13). Erythropoietin expression in HEK293 cells was measured using the Simple Plex™ Human Erythropoietin Cartridge on an ELLA™ instrument (Figure 13). Regardless of the helper lipid used in the LNP composition, LB#107 protected the activity of the LNPs (Figure 13A, LB#107 for LNPs made with DSPC, DPPC, DOPE, and DOPC; Figure 13B, LB#107 and LB#110 for LNPs made with DSPC; Figure 13C, LB#107 and LB#189 for LNPs made with DPPC).

[0118] The corresponding encapsulation efficiencies (%) of the reconstituted cakes after storing the cakes for 7 days at the indicated temperatures are shown in Table 8. Freeze-dried cakes of fresh LNP (no lyophilization) and base composition (BC) at each temperature are shown as positive controls.

[0119] [Table 8] [Example]

[0120] This example demonstrates the effectiveness of a lyophilization buffer to protect mRNA-LNPs encoding luciferase protein.

[0121] Expression of firefly luciferase protein in HEK cells: HEK 293 cells (ATCC) were cultured at a density of 12 × 10 in 100 μL of complete DMEM (Gibco). 3 Cells were seeded at 1 cell / well in white 96-well plates and grown for 24 hours at 37°C with 5% CO2. Lyophilized mRNA LNPs encoding firefly luciferase protein were reconstituted in 1x PBS and added to the cells at doses ranging from 50 ng to 100 ng, along with other controls. After 24 hours of treatment, cells were analyzed for cell viability, and luciferase expression was measured using the ONE-Glo™ + Tox Assay Kit (Promega) according to the manufacturer's protocol.

[0122] Lyophilization: Fluc mRNA-LNPs were prepared using PNI 516 and 127 in a VB composition in N / P-8 and lyophilized by mixing with LB#107. The lyo-cakes were stored at RT for 1 week and reconstituted in 1x PBS. The control LNPs were BC-treated LNPs (BC, -80°C) stored at -80°C. The results are shown in Figure 14. In the figure, LB#107 showed similar or better activity with both lipids (PNI 516 and 127) in expressing luciferase protein in HEK cells compared with the control BC stored at -80°C. Table 9 shows that LB#107 performed well in protecting the size and EE of lyophilized cakes of PNI 516 and 127 LNPs encoding luciferase protein mRNA stored at RT for 1 week.

[0123] [Table 9] [Example]

[0124] This example demonstrates that the lyophilization buffer of the present invention maintained the activity of mRNA-LNPs in mice: EPO expression in C57BL / 6 mice. Reconstituted lyophilized LNPs (cakes stored at -20°C, 4°C, and RT for 7 days) containing EPO-encoding mRNA and fresh LNPs were administered to mice. All formulations were made using a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG composition containing PNI 516. Blood was collected from the tail vein, and serum was prepared according to standard procedures. A 6-hour serum analysis of EPO expression from mouse serum was assessed by automated ELISA using the ELLA Simple Plex™ Human Erythropoietin Cartridge kit (Protein Simple™, Biotech).

[0125] EPO levels in mice are shown in Figure 15. LB#107 was the best performing candidate tested at RT and 4°C, while LB#189 performed best at -20°C. Freshly prepared LNPs stored in PBS at 4°C were used as controls. Regardless of the 7-day time effect at various storage temperatures, all of the reconstituted lyophilized LNPs showed similar or better efficacy compared to the fresh LNP control. Encapsulation efficiency is shown in Figure 16. LB#107, 110, and 189 retained EE after lyophilization. The size and PDI of these same LNPs are shown in Figure 17. There was no substantial effect on size and PDI as a result of lyophilization compared to the control. [Example]

[0126] This example demonstrates that the lyophilization buffer of the present invention protects plasmid-encapsulated LNPs. Custom-made CMV-eGFP-pDNA plasmid and PN1516 lipids by GenScript USA, Piscataway, NJ, were used in the LNP formulation. LNP preparation is described above. The plasmid was formulated at an N / P ratio of 8 using a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG composition containing PN1516. LNP samples were then lyophilized, and the lyocakes were stored at RT for 1 week. After reconstitution in 1x PBS, size and EE were measured. As shown in Table 10, LB#107, 110, and 189 protected the size and PDI of LNPs. All tested LNPs had a mean diameter of 93-115 nm, a PDI of 0.2, and an encapsulation efficiency of greater than 80% before and after lyophilization and reconstitution.

[0127] [Table 10] [Example]

[0128] This example demonstrates that the lyophilization buffer of the present invention protects eGFP mRNA. LNPs were prepared as described in Example 2 using PNI 516 and lipid composition VA. LNPs were lyophilized as described in Example 5 using lyophilization buffer (LB) #107, 110, 163, 164, 167, 168, 170, 178, 179, 180, 181, and 189. The lyo-cakes were stored at RT for 24 hours and reconstituted in 1x PBS before performing measurements on the lyophilized LNPs. Each of the lyo-buffers protected the size and EE of PNI 516 LNPs encoding eGFP mRNA. The lyo-buffer LNPs exhibited similar average size, PDI, and EE to fresh LNPs (Table 7b).

[0129] [Table 11] [Example]

[0130] This example demonstrates a method for selecting lyophilized reagents to protect LNPs. LNPs containing PN1516 47.5 mol% / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG and encapsulating mRNA encoding the SARS-CoV-2 full-length spike protein were prepared as described above. Lyo buffers #101, 109, 112, 113, 119, 120, 134, 135, 136, 140, 142, 143, 147, 148, 157, 161, 162, 166, 171, 182, and 187 (listed in Table 3 above) were tested for their ability to maintain the in vitro potency of saRNA LNPs. Lyophilized cakes were then reconstituted with 1x PBS, sterilized, analyzed, and used for cell therapy.

[0131] Western blot: HEK-293 cells were seeded (0.3 × 10 ) in 6-well plates for 2 days. 6The cells were then treated with 1 μg / mL of lyophilized LNPs (1000 cells / well) for 1 day. Cell lysates were prepared using IP lysis buffer. Protein quantification was performed using a BCA kit according to the supplier's protocol (Pierce™ BCA Protein Assay Kit). Protein samples were denatured using loading dye and 2-mercaptoethanol. SDS-PAGE was performed in a Mini Gel Tank (175 V, 500 mA, 90 min). The gel was transferred to a nitrocellulose membrane (Thermo Fisher) using an iBlot™ 2 device (Thermo Fisher). After blocking, the membrane was stained with primary antibody for 2 hours at RT on a shaker, followed by secondary antibody staining. The blot was developed using a chemiluminescent substrate. The blot was imaged using an iBright instrument (Thermo Fisher). Spike protein expression was qualitatively determined, and the performance of the lyophilized LNPs was assessed compared to the control. Figure 18 shows the results of Western blot analysis of protein expression obtained from cells treated with NALNPs freeze-dried using 31 LB candidates. Protein samples obtained from LNPs freeze-dried using LBs #54, 120, 134, 135, 148, 157, 167, 168, 187, and 208 showed significant activity after freeze-drying. [Example]

[0132] Table 12 shows the encapsulation efficiency (EE) of LNPs containing PNI 516 47.5 mol% / 12.5 mol% DSPC / 38.5 mol% cholesterol / 1.5 mol% PEG-DMG composition encapsulating A3 saRNA after 12 days of cake storage at RT. LNPs lyophilized using LB#120, 163, 166, and 167 yielded higher EE% compared to LB#54.

[0133] [Table 12] [Example]

[0134] In contrast to LB#54, the mRNA LNP-containing leads LB#108, LB#167, LB#218, and LB#241 exhibited sizes below 100 nm, a desirable quality characteristic for parenteral injection. In Table 13, the size of LNPs containing IL PN1 516 and hEPO mRNA using a 40 mol% IL / 12.5 mol% DSPC / 46.0 mol% cholesterol / 1.5 mol% PEG-DMG composition showed significant advantages over PVA LB#54 after 2-month cake storage at 4°C.

[0135] [Table 13] [Example]

[0136] The size change of LNPs containing IL PN1 516 and A5 saRNA using the above composition was studied after storing the cakes for 1 month at 4° C. Leads LB#108, LB#162, LB#164, and LB#167 containing A5 saRNA LNPs showed a size of less than 100 nm, a desirable quality characteristic for parenteral injection.

[0137] [Table 14] [Example]

[0138] In vivo EPO expression This example describes the procedure used to evaluate hEPO protein expression in vivo from hEPO mRNA-LNPs (reconstituted after 2-month storage of the cake at 4°C). LNPs were intramuscularly injected into mice (6-week-old male BALB / c mice) at a dose of 0.25 mg / kg (5 μg / 20 g mouse). Serum samples were collected 6 and 24 hours after injection. For serum preparation, whole blood was collected and allowed to clot by leaving the collection tube at room temperature for 15–30 minutes. The clot was removed by centrifuging the tube at 1000–2000 × g for 10 minutes at 4°C. The clear, yellow-orange supernatant was carefully removed and transferred to a sterile, screw-capped, clear polypropylene tube on ice. The serum was then stored at -80°C until further use. SARS-CoV-2 antigen-specific IgG levels in the serum were determined using an enzyme-linked immunosorbent assay (ELISA). Leads LB#108, LB#164, and LB#167 showed better activity than LB#54. Leads LB#108, LB#167, LB#218, LB#241, and LB#260 retained activity compared to PVA containing LB#54 (Figure 19). [Example]

[0139] In vivo vaccine expression This example describes the procedure used for in vivo evaluation of SARS-CoV-2 expression of SARS-CoV-2-expressing A5 PN1 saRNA LNPs, reconstituted after one month of cake storage at 4°C. On day 0, LNPs were intramuscularly injected into mice (6-week-old male BALB / c mice) at a dose of 0.05 mg / kg (1 μg / 20 g mouse). Serum samples were collected 21 days after injection. For serum preparation, blood was allowed to clot for 15–30 minutes at room temperature. Clots were removed by centrifuging the tubes at 1000–2000 × g for 10 minutes at 4°C. The clear, yellow-orange supernatant was carefully removed and transferred to a sterile, screw-capped, clear polypropylene tube on ice. The serum was then stored at -80°C until further use. SARS-CoV-2 antigen-specific IgG levels in the serum were determined using an enzyme-linked immunosorbent assay (ELISA). As shown in Figure 20, leads LB#108, LB#164 and LB#167 showed better activity than LB#54.

[0140] In the context of describing the present invention (particularly in the context of the claims below), use of the terms "a," "an," "the," and "at least one" and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" following a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item (A or B) selected from the listed items or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise indicated. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method for individually referencing each individual value within the range, and each individual value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any examples provided herein, or the use of typical language (e.g., "such as"), are intended merely to further clarify the invention and do not limit the scope of the invention unless specifically claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0141] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect that skilled artisans will utilize such variations as necessary, and the inventors intend the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context.

[0142] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

Claims

1. (a) lipid nanoparticles containing nucleic acids, and (b) a lyophilization buffer solution comprising a sugar and a lyophilization reagent selected from a sugar-mimetic oligomer / polymer, an amphiphilic thermoresponsive polymer, an ethylene glycol-mimetic polymer, a hydrophilic monomer, and a hydrophilic polymer; Lyophilized nucleic acid lipid nanoparticles (NALNPs) comprising:

2. 2. The lyophilization buffer of claim 1, wherein the sugar-mimetic oligomer / polymer is n-octanoylsucrose, cyclodextrin, beta-cyclodextrin polymer, dextran, trehalose, sorbitol-core carboxy-terminated PEG, Betadex™ sulfobutyl ether sodium, or 2-hydroxypropyl-beta-cyclodextrin.

3. 2. The lyophilization buffer of claim 1, wherein the amphiphilic thermoresponsive polymer is poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide).

4. 2. The lyophilization buffer of claim 1, wherein the ethylene glycol-mimetic polymer is a 6-arm branched PEG, a 5-arm branched PEG, a 3-arm branched PEG, trimethylpropane ethoxylate, polyethylene glycol, poloxamer 407, an amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol).

5. 2. The lyophilization buffer of claim 1, wherein the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), an amino acid, or L-arginine.

6. The NALNP of any one of claims 1 to 5, wherein the mass / mass ratio of the lyophilization reagent to the sugar is 1:

8.

7. The NALNP of any one of claims 1 to 5, wherein the mass / mass ratio of the lyophilization reagent to the sugar is 1:

5.

8. 10. The NALNP of claim 1, further comprising a pharmaceutically acceptable diluent selected from Tris, sodium acetate, sodium citrate, dextrose, and saline solution.

9. 8. The NALNP of claim 1, 6, or 7, wherein the NALNP has a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

10. 2. The NALNP of claim 1, wherein the sugar is sucrose.

11. The NALNP of any one of claims 1 to 8, wherein the NALNP is in an anhydrous form.

12. (a) mixing a nucleic acid with a lipid mixture solution containing an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles; (b) combining the lipid nanoparticles obtained in (a) with a lyophilization buffer containing a sugar and a lyophilization reagent selected from the group consisting of polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-beta-cyclodextrin, N,N-dimethylacrylamide, poly(N,N-diethylacrylamide), poly(2-ethyl-2-oxazoline), glycerol ethoxylate, amine-terminated 4-arm PEG, 6-arm branched PEG, 5-arm branched PEG, and sorbitol-core carboxy-terminated PEG, and a pharmaceutically acceptable diluent; (c) lyophilizing the combination of lipid nanoparticles and lyophilization buffer obtained in step (b) to obtain anhydrous lyophilized NALNPs. A method for preparing freeze-dried NALNP, comprising:

13. lyophilizing the combination of nanoparticles and a lyophilization buffer; (a) freezing the combination of lipid nanoparticles and lyophilization buffer at −40 to −90° C. for 60 to 400 minutes; (b) drying the combination of lipid nanoparticles and lyophilization buffer at −20 to −40° C. and 30 to 100 mTorr for 700 to 980 minutes; (c) drying the combination of lipid nanoparticles and lyophilization buffer at 4-10°C and 30-100 mTorr for 250-500 minutes; 13. The method of claim 12, comprising:

14. 13. The method of claim 12, wherein the lyophilization buffer comprises a pharmaceutically acceptable diluent.

15. 15. The method of claim 14, wherein the pharmaceutically acceptable diluent is Tris, sodium acetate, sodium citrate, dextrose, saline, or water.

16. 16. The method of any one of claims 10 to 15, wherein the lyophilization buffer has a mass / mass ratio of lyophilization reagent to sugar to pharmaceutically acceptable diluent of 1:4:40 to 1:8:

80.

17. 16. The method of any one of claims 10-15, wherein the NALNP has a mass / mass ratio of nucleic acid to lyophilized reagent to sugar of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

18. 16. The method of any one of claims 10 to 15, wherein the sugar is sucrose.

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