Lipid nanoparticle lyophilization methods and compositions

Abstract

The present invention provides a lyophilized nucleic acid lipid nanoparticle (NALNP) comprising (a) a lipid nanoparticle comprising a nucleic acid, and (b) a lyophilization buffer comprising a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent; and a preparation method thereof.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Patent Application 18 / 479,482, filed on December 12, 2022, under 35 USC § 119(e). Background of the Invention

[0004] Due to their short production time, mRNA vaccines are well - suited for rapid response to new threats, such as the COVID - 2019 virus. Additionally, nucleic - acid - based vaccines are superior to traditional vaccines in terms of safety and efficacy. Messenger RNA (“mRNA”) vaccines are superior to DNA - based vaccines, which need to cross the nuclear membrane to function and carry the risk of integration into the host genome. Messenger RNA vaccines are degraded by exonucleases and endonucleases in vivo in the absence of a delivery system, so mRNA vaccines require a carrier. Currently, lipid nanoparticles (LNPs) are one of the most commonly used carriers for in - vivo RNA delivery. Lipid nanoparticles or LNPs typically consist of a lipid or aqueous core surrounded by a lipid bilayer shell made of a combination of different lipids, each with a different function.

[0005] Current mRNA vaccines must be stored frozen at low temperatures for storage. This requirement limits their distribution. Thus, there is still a need for suitable lyophilization protocols to maintain mRNA vaccines in a stable and effective form.

[0006] Summary of the Invention

[0007] According to one embodiment, there is provided a lyophilized nucleic acid lipid nanoparticle (NALNP) comprising:

[0008] (a) a lipid nanoparticle comprising a nucleic acid; and

[0009] (b) a lyophilization buffer comprising a sugar and a lyophilization reagent selected from sugar - mimicking oligomers / polymers, amphiphilic thermoresponsive polymers, ethylene - glycol - mimicking polymers, hydrophilic monomers, and hydrophilic polymers.

[0010] In some embodiments, the sugar - mimicking oligomer / polymer is octanoyl sucrose, cyclodextrin, β - cyclodextrin polymer, dextran, trehalose, carboxyl - terminated PEG with a sorbitol core, Betadex TM sodium sulfobutyl ether or 2 - hydroxypropyl - β - cyclodextrin.

[0011] In other embodiments, the amphiphilic thermoresponsive polymer is poly(N - vinylcaprolactam), poly(N,N - dimethylacrylamide), poly(N,N - diethylacrylamide), or poly(acrylamide).

[0012] In other embodiments, the ethylene glycol mimicking polymer is 6-arm branched PEG, 5-arm branched PEG, 3-arm branched PEG, trimethylolpropane ethoxylate, polyethylene glycol, poloxamer 407, amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol).

[0013] In still other embodiments, the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, diglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), an amino acid, or L-arginine.

[0014] In an embodiment of the present invention, the wt / wt ratio of the lyophilized reagent to the sugar is 1:8 or 1:4. In an embodiment, the pharmaceutically acceptable diluent is selected from solutions of Tris, sodium acetate, sodium citrate, glucose, and saline. In an embodiment, the nucleic acid of the NALNP and the lyophilized reagent and the sugar have a wt / wt ratio of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000. In an embodiment, the sugar is sucrose. In many embodiments, the NALNP is in an anhydrous form.

[0015] According to an embodiment, a method for preparing a lyophilized NALNP is provided, comprising

[0016] (a) mixing a nucleic acid with a lipid mixture solution comprising an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles;

[0017] (b) combining the lipid nanoparticles obtained in (a) with a lyophilization buffer comprising a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent, the lyophilization reagent being selected from polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-β-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 carboxyl-terminated PEG with a sorbitol core; and

[0018] (c) lyophilizing the combined lipid nanoparticles and lyophilization buffer obtained in (b) to obtain anhydrous lyophilized NALNP.

[0019] In an embodiment, lyophilizing the combined nanoparticles and lyophilization buffer comprises:

[0020] (a) freezing the combined lipid nanoparticles and lyophilization buffer at -40 to -90 °C for 60 - 400 minutes,

[0021] (b) Dry the combined lipid nanoparticles and lyophilization buffer at -20 to -40 °C and 30 - 100 mTorr for 700 - 980 minutes, and

[0022] (c) Dry the combined lipid nanoparticles and lyophilization buffer at 4 - 10 °C at 30 - 100 mTorr for 250 - 500 minutes. In an embodiment, the lyophilization buffer contains a pharmaceutically acceptable diluent.

[0023] In an embodiment, the pharmaceutically acceptable diluent is Tris, sodium acetate, sodium citrate, glucose, saline, or water. In some embodiments, the weight - to - weight ratio of the lyophilization reagent, sugar, and pharmaceutically acceptable diluent in the lyophilization buffer is between 1:4:40 and 1:8:80. In an embodiment, the weight - to - weight ratio of the nucleic acid of the NALNP, lyophilization reagent, and sugar is 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0024] In an embodiment, the sugar is sucrose.

[0025] The features and advantages of the subject matter here become more apparent from the following detailed description of the selected embodiments as shown in the accompanying drawings. As will be realized, the disclosed and claimed subject matter can be modified in various aspects, all of which do not depart from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature and not restrictive, and the full scope of the subject matter is set forth in the claims. Brief Description of the Drawings

[0027] Figure 1 is a diagram of an exemplary lyophilization process according to the present invention;

[0028] Figure 2 is a graph showing the size and polydispersity results of lyophilized LNP formulated with two different saRNAs (SARS - CoV - 2 spike protein - specific A3b or A3p antigens) at N / P - 8 in a VA composition and reconstituted in 1X phosphate - buffered saline (PBS) after storage at room temperature (RT) for 24 hours (hr);

[0029] Figure 3 is a graph showing the encapsulation efficiency results of lyophilized LNP made with two different saRNAs (SARS - CoV - 2 spike protein - specific A3b or A3p antigens) at N / P - 8 in a VA composition, using lyophilization buffers (LB) #107 and 110, stored at RT for 24 hours, and reconstituted in 1X PBS;

[0030] Figure 4Shows the Western blot results of SARS-CoV-2 spike protein expressed in vitro in HEK-293 cells, which were treated with freeze-dried LNP containing PNI 516 with saRNA at 1 μg / mL for 24 hours. The freeze-dried LNP was stored at 4 °C for 24 hours using LB#107, 110, 163, 164, 168, 178, 179, 180, 181, 182, 186 and 189 and reconstituted in 1XPBS;

[0031] Figure 5 Is a Western blot image showing the expression of SARS-CoV-2 spike protein in HEK293 cells, which were treated with freeze-dried PNI 516 LNP encapsulating SARS-CoV-2 spike protein-specific A3saRNA of VB composition (N / P-8). The LNP was freeze-dried and stored at -20 °C and 4 °C for one week and then reconstituted;

[0032] Figure 6 Shows the expression of SARS-CoV-2 spike protein in HEK293 cells determined by Western blot analysis. LNP containing PNI 516 and SARS-CoV-2 spike protein-specific A3saRNA was formulated in VB composition at N / P-8, freeze-dried, stored at RT for one week and then reconstituted;

[0033] Figure 7 Shows the results of Western blot analysis of SARS-CoV-2-specific spike protein expression in HEK 293 cells after treatment with freeze-dried LNP with saRNA at a concentration of 0.25 μg / mL. The LNP was formulated with PNI 516 and SARS-CoV-2 spike protein-specific A5 saRNA at N / P-8 in VB composition. After freeze-drying, the freeze-dried cake was stored at RT and 4 °C for three months and reconstituted in 1XPBS;

[0034] Figure 8 Shows a graph of SARS-CoV-2 spike protein-specific IgG expression in C57BL / 6 mice on day 42 after intramuscular administration of 1 μg / mouse dose of SARS-CoV-2 spike protein-encoding saRNA LNP after freeze-drying, storage at three different temperatures (-20 °C, 4 °C and RT) for one week and reconstitution. The LNP contained PNI 516 according to VB composition (N / P-8);

[0035] Figure 9 Is a graph showing the expression of human erythropoietin (hEPO) protein in HEK293 cells treated with freeze-dried LNP at a dose of 1 μg / mL, which was stored at three different temperatures (20 °C, 4 °C and RT) for one week and reconstituted in 1X PBS;

[0036] Figure 10 A-10E is a graph showing the EPO protein expression levels in HEK293 cells treated with freeze-dried and reconstituted LNPs at a dose of 1 μg / mL in 1XPBS, where the LNPs were stored at RT for one week. These EPO mRNA-LNPs contained PNI 516 in the VB composition at N / P-8 ( Figure 10 A), PNI 127 ( Figure 10 B), dilinoleyl methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) ( Figure 10 C), 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) ( Figure 10 D), or 4-(dimethylamino)-9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl butyrate (BOCHD-C3-DMA) (

[0037] E); Figure 10 E);

[0038] Figure 11 A is a graph showing the encapsulation efficiency results of freeze-dried EPO mRNA-LNPs reconstituted in 1XPBS formulated with PNI 516 and stored at RT for 1 week;

[0039] Figure 11 B is a graph showing the encapsulation efficiency results of freeze-dried EPO mRNA-LNPs reconstituted in 1X PBS formulated with PNI 127 and stored at RT for 1 week;

[0040] Figure 11 C is a graph showing the encapsulation efficiency results of freeze-dried EPO mRNA-LNPs reconstituted in 1XPBS formulated with dilinoleyl methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and stored at RT for 1 week;

[0041] Figure 11 D is a graph showing the encapsulation efficiency results of freeze-dried EPO mRNA-LNPs reconstituted in 1X PBS formulated with 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) and stored at RT for 1 week;

[0042] Figure 11 E is a graph showing the encapsulation efficiency results of freeze-dried EPO mRNA-LNPs reconstituted in 1XPBS formulated with 4-(dimethylamino)-9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl butyrate (BOCHD-C3-DMA) and stored at RT for 1 week;

[0043] Figure 12 Panel A shows the size and polydispersity results of lyophilized EPO mRNA-LNP formulated with PNI 516 and reconstituted in 1X PBS after storage at RT for one week;

[0044] Figure 12 Panel B shows the size and polydispersity results of lyophilized EPO mRNA-LNP formulated with PNI 127 and reconstituted in 1X PBS after storage at RT for one week;

[0045] Figure 12 Panel C shows the size and polydispersity results of lyophilized EPO mRNA-LNP formulated with dimethylaminobutyric acid dilinoleyl methyl ester (DLin-MC3-DMA) and reconstituted in 1X PBS after storage at RT for one week;

[0046] Figure 12 Panel D shows the size and polydispersity results of lyophilized EPO mRNA-LNP formulated with 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) and reconstituted in 1X PBS after storage at RT for one week;

[0047] Figure 12 Panel E shows the size and polydispersity results of lyophilized EPO mRNA-LNP formulated with 4-(dimethylamino)butanoic acid, 9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester (BOCHD-C3-DMA) and reconstituted in 1X PBS after storage at RT for one week;

[0048] Figure 13 Panel A shows the EPO protein expression levels in HEK293 cells treated with LNP lyophilized and reconstituted in 1X PBS after storage at RT for one week at a dose of 1 μg / mL. The LNP of PNI 516 and EPO mRNA-LNP was lyophilized with LB#107 buffer according to the VB composition (N / P - 8) and varied with different co-lipids DSPC, DPPC, DOPE or DOPC;

[0049] Figure 13 Panel B shows the EPO protein expression levels in HEK293 cells treated with LNP lyophilized and reconstituted in 1X PBS after storage at RT for one week at a dose of 1 μg / mL. The LNP of PNI 516, EPO mRNA-LNP and co-lipid DSPC was lyophilized with LB#110 and LB#189 buffers;

[0050] Figure 13Panel C shows the EPO protein expression levels in HEK293 cells treated with LNP that was lyophilized at a dose of 1 μg / mL and stored at RT for one week before being reconstituted in 1X PBS. The LNP of PNI 516 and EPO mRNA-LNP, along with the helper lipid DPPC, was lyophilized using LB#107 and LB#189 buffers;

[0051] Figure 14 is a panel showing the luciferase protein expression levels in HEK293 cells treated with LNP containing PNI 516 or PNI 127 that was lyophilized at a dose of 25 ng / well and stored at RT for one week before being reconstituted in 1X PBS;

[0052] Figure 15 is a panel showing the EPO expression levels in C57BL / 6 mice after i.v. administration of a dose of 0.25 mg / Kg of PNI 516 and recombinant human EPO-encoding mRNA LNP (VB formulation, N / P - 8), where the LNP was lyophilized and stored at three different temperatures (-20 °C, 4 °C, and RT) for one week before being reconstituted in 1X PBS prior to administration;

[0053] Figure 16 is a panel showing the encapsulation efficiency results of lyophilized LNP made from PNI 516 and EPO-encoding mRNA using 3 different lyophilization buffers. 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;

[0054] Figure 17 is a panel showing the size and PDI results of lyophilized LNP made from PNI 516 and EPO-encoding mRNA using 3 different lyophilization buffers. 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;

[0055] Figure 18Photographic image of a Western blot of a cell lysate of HEK-293 cells treated with freeze-dried LNP containing in vitro-expressed SARS-CoV-2 spike protein mRNA (0.25 μg / mL saRNA for 24 hours), the LNP freeze-dried with a freeze-drying 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 LNP stored at RT for 12 days and reconstituted in 1X PBS before treatment;

[0056] Figure 19 Graphical representation of EPO protein levels in mouse serum treated with LNP lyoprotected (LB#54, 108, 167, 218, 241, and 260) after storage at 4°C for 2 months; and

[0057] Figure 20 Graphical representation of SARS-CoV-2 spike protein-specific IgG levels in mouse serum treated with a dosing material containing freeze-drying buffer leaders LB#354, 108, 164, and 167 after LNP storage at 4°C for 1 month. DETAILED DESCRIPTION OF THE INVENTION

[0059] The freeze-dried nucleic acid lipid nanoparticles (NALNPs) of the present invention comprise (a) lipid nanoparticles comprising nucleic acids, and (b) a freeze-drying buffer comprising sugars, freeze-drying reagents, and pharmaceutically acceptable diluents.

[0060] The freeze-dried NALNPs of the present invention comprise lipid nanoparticles comprising nucleic acids. Lipid nanoparticles are a subgroup of lipid particles with 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 diameter of the lipid particles is about 300 nm or less, 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 diameter of the lipid particles is about 50 to about 150 nm. Smaller particles generally exhibit increased circulation lifetimes in vivo compared to larger particles. Smaller particles have an increased ability to reach the tumor site compared to larger nanoparticles. In one embodiment, the diameter of the lipid particles is about 15 to about 50 nm.

[0061] Lipid nanoparticles are generally spherical assemblies of lipids, nucleic acids, sterols, and stabilizers. The positive and negative charges, ratios, and hydrophilic and hydrophobic properties determine the physical structure of the lipid particles in terms of size and component orientation. The structural organization of these lipids can result in an aqueous interior with a minimal bilayer (such as in liposomes), or it may have a solid interior (such as in solid nucleic acid lipid nanoparticles). There may be single or multiple forms of phospholipid monolayers or bilayers. The size of the lipid particles is between 1 and 1000 μm.

[0062] In some embodiments, the lipid nanoparticle comprises a lipid mixture solution and a nucleic acid. In some embodiments, the lipid mixture solution comprises an ionizable lipid, a structural lipid, a sterol, and a stabilizer. "N / P" is the ratio of the number of moles of the amine groups of the ionizable lipid to the number of moles of the phosphate groups of the nucleic acid. In some embodiments, the N / P ratio is 4 - 12. In a preferred embodiment, the N / P ratio is 6 - 10. For example, in a preferred embodiment, the N / P ratio is 6, 8, or 10. The nucleic acid associates with the lipid mixture composition to form an LNP at a predetermined ratio, such as the ionizable lipid amine (N) and nucleic acid phosphate (P) are quantified as N / P 4, N / P 6, N / P 8, N / P 10, N / P 12, or any other suitable N / P ratio.

[0063] In some embodiments, the lipid mixture solution comprises a stabilizing agent or a stabilizer. Any suitable stabilizing agent or stabilizer can be used in the embodiments of the present invention. In some embodiments, the stabilizer is selected from polysorbate (Tween), Brij TM S20 (polyoxyethylene (20) stearyl ether), Brij TM 35 (polyoxyethylene lauryl ether, polyethylene glycol lauryl ether), Brij TM S10 (polyethylene glycol octadecyl ether, polyoxyethylene (10) stearyl ether), Myrj TM52 (polyoxyethylene (40) stearate), PEG-DMG, PEG-DMG 2000, glyceryl trioleate, tridecyl-D-maltoside, Tween 20, polysorbate 80, lipid H, TPGS1000, polyoxyethylene (4) lauryl ether, and DiD. Stabilizer combinations are also used in some embodiments, including polysorbates and maltosides, alkylpolyglycosides (TBD), PEG-conjugated lipids, or other polymer-conjugated lipids. In some embodiments, the lipid mixture solution contains more than one stabilizing agent or stabilizer. For example, in some embodiments, the lipid mixture solution contains one or more, two or more, three or more, or four or more stabilizing agents or stabilizers.

[0064] In some embodiments, the lipid mixture solution contains ionizable lipids. Any suitable ionizable lipid can be used in the embodiments of the present invention. An ionizable lipid is a lipid that is cationic or becomes ionizable (protonated) when the pH is lowered below the pKa of the ionizable group of the lipid, but is more neutral at higher pHs. At a pH below the pKa, the lipid is capable of associating with negatively charged nucleic acids (such as oligonucleotides). Examples of suitable ionizable lipids are found in PCT Publication Nos. WO2020252589 and WO2021000041.

[0065] In some embodiments, the ionizable lipid is DODMA (1,2-dioleoyl-3-dimethylaminopropane), DLin-MC3-DMA (O-(Z,Z,Z,Z-nonatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino)), DLin-KC2-DMA (2-dilinoleoyl-4-dimethylaminoethyl-[1,3]-dioxolane), butyric 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)heptadec-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butyrate (referred to as PNI 516) (WO2020 / 252589) or (2R,3S,4R)-2-((((1,4-dimethylpiperidin-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl (9E,9'E,12E,12'E)-bis(octadec-9,12-dienoate) (referred to as PNI 127) (WO 2021 / 000041).

[0066] In some embodiments, the lipid mixture solution comprises a structural lipid. Structural lipids may also be referred to as helper lipids or neutral lipids. Any suitable structural lipid can be used in the embodiments of the present invention. Suitable structural lipids support the formation of particles during the manufacturing process. A structural lipid refers to any one of a variety of lipid species that exist in anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative structural lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, diacyl phosphatidylglycerol, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

[0067] Exemplary structural lipids include zwitterionic lipids such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), palmitoyl oleoyl phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In a preferred embodiment, the structural lipid is distearoyl phosphatidylcholine (DSPC).

[0068] In another embodiment, the structural lipid is any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol such as dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl oleoyl phosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacyl phosphatidylserine, diacyl phosphatidic acid, and other anionic modifying groups linked to neutral lipids. Other suitable structural lipids include glycolipids (e.g., monosialoganglioside GM1).

[0069] In some embodiments, the lipid mixture solution comprises a sterol. Any suitable sterol can be used. In some embodiments, the sterol is cholesterol, β-sitosterol, 20-α-hydroxy sterol, or phytosterol. In a preferred embodiment, the sterol is cholesterol.

[0070] The lipid mixture can comprise any suitable combination of ionizable lipids, structural lipids, sterols, and stabilizers. In some embodiments, the lipid mixture comprises 47.5 mol% ionizable lipid, 12.5 mol% structural lipid, 38.5 mol% sterol, and 1.5 mol% stabilizer. In a preferred embodiment, the lipid mixture comprises 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 comprises 47.5 mol% IL, 122.5 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG. In other embodiments, the lipid mixture comprises 40 mol% ionizable lipid, 20 mol% structural lipid, 37.5 mol% sterol, and 2.5 mol% stabilizer. 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 TM S10.

[0071] The lipid nanoparticles comprise nucleic acid. Any suitable nucleic acid can be used in the lipid nanoparticles. Nucleic acid is a substance that is intended to have a direct effect on the diagnosis, cure, mitigation, treatment, or prevention of disease, or on the restoration, correction, or modification of physiological function, or 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 a single gene. In some embodiments, the nucleic acid is referred to as a nucleic acid therapeutic or NAT.

[0072] The lyophilized NALNP of the present invention comprises a lyophilization buffer (sometimes referred to as a lyo buffer) that contains sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent.

[0073] The lyophilization buffer of the present invention contains sugar. Any suitable sugar can 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 contains more than one type of sugar. In some embodiments, the lyophilization buffer contains one or more, two or more, or three or more types of sugar.

[0074] The lyophilized buffer of the present invention contains a lyophilized reagent. In some embodiments, the lyophilized reagent is selected from polyvinyl alcohol, sugar-mimicking oligomers / polymers, amphiphilic thermoresponsive polymers, ethylene glycol-mimicking polymers, or hydrophilic monomers or polymers. In some embodiments, the sugar-mimicking oligomers / polymers are octanoyl sucrose, cyclodextrin, β-cyclodextrin, dextran, trehalose, carboxyl-terminated PEG with a sorbitol core, Betadex TM sodium sulfobutyl ether, or 2-hydroxypropyl-β-cyclodextrin. In some embodiments, the amphiphilic thermoresponsive polymers are poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide). In some embodiments, the ethylene glycol-mimicking polymers are 6-arm branched PEG, 5-arm branched PEG, 3-arm branched PEG, trimethylolpropane ethoxylate, polyethylene glycol, Pluronic TM (F-127), amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol). In some embodiments, the hydrophilic monomers or polymers are propylene glycol, glycerol, polypropylene glycol, triglycerol, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), amino acids, or L-arginine.

[0075] In a preferred embodiment, the lyophilized reagent is polyvinylpyrrolidone. In another preferred embodiment, the lyophilized reagent is 2-hydroxypropyl-β-cyclodextrin.

[0076] The lyophilized buffer of the present invention contains a pharmaceutically acceptable diluent. Any suitable pharmaceutically acceptable diluent can be used. In some embodiments, the pharmaceutically acceptable diluent is selected from Tris, sodium acetate, glucose, 5% glucose, saline, PBS, lactated Ringer's solution, 5% human serum albumin, and solutions of water. In a preferred embodiment, the pharmaceutically acceptable diluent is Tris buffer. In some embodiments, the lyophilized buffer contains more than one pharmaceutically acceptable diluent. For example, in some embodiments, the lyophilized buffer contains one or more, two or more, or three or more pharmaceutically acceptable diluents. In a preferred embodiment, the lyophilized buffer contains Tris buffer and PBS. In some embodiments, the salt concentration of the pharmaceutically acceptable diluent is 0 - 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 the salt concentration is between any two of the above values.

[0077] 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% (w / v) sucrose (10 mg / mL) dissolved in 20 mM Tris buffer and 1X PBS. In some embodiments, the lyophilization buffer comprises a base composition buffer and a lyophilization reagent. In a preferred embodiment, the lyophilization buffer comprises a lyophilization reagent in an amount of 1-5% w / v. For example, in some embodiments, the lyophilization buffer comprises a lyophilization reagent in an amount of 1% w / v, 1.5% w / v, 2% w / v, 2.5% w / v, 3% w / v, 3.5% w / v, 4% w / v, 4.5% w / v, 5% w / v, or % w / v in an amount between any two of the foregoing values.

[0078] In some embodiments, the wt / wt ratio of the lyophilization reagent to the sugar in the lyophilization buffer is between 1:1 and 1:20. For example, in some embodiments, the wt / wt ratio of the lyophilization reagent to the sugar in the lyophilization buffer is 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 the wt / wt ratio of the lyophilization reagent to the sugar is between any two of the foregoing values. In a preferred embodiment, the wt / wt ratio of the lyophilization reagent to the sugar in the lyophilization buffer is between 1:4 and 1:10. In another embodiment, the wt / wt ratio of the lyophilization reagent to the sugar is 1:8.

[0079] In some embodiments, the wt / wt ratio of the lyophilized reagent of the lyophilization buffer, sugar, and pharmaceutically acceptable diluent is between 1:1:40 and 1:10:80. For example, in some embodiments, the wt / wt ratio of the lyophilized reagent, sugar, and 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 above values. In other embodiments, the wt / wt ratio of the lyophilized reagent, sugar, and 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 above values. In other embodiments, the wt / wt ratio of the lyophilized reagent, sugar, and 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 above values.

[0080] In other embodiments, the wt / wt ratio of the lyophilized reagent, sugar, and 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 above values. In a preferred embodiment, the wt / wt ratio of the lyophilized reagent, sugar, and pharmaceutically acceptable diluent of the lyophilization buffer is between 1:4:40 and 1:8:80.

[0081] The term "diluent" in this application refers to liquid or lyophilized form. Thus, in lyophilized form, the "diluent" is the dehydrated residue of the diluent used during the lyophilization process.

[0082] In some embodiments, the wt / wt ratio of the nucleic acid of NALNP, lyophilized reagent, and sugar is 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0083] In some embodiments, NALNP is in anhydrous form. NALNP is in anhydrous form after lyophilization as described herein. In some embodiments, NALNP is in anhydrous form and consists of a lyophilized or lyo cake. In some examples, NALNP is in reconstituted form. In reconstituted form, a pharmaceutically acceptable diluent has been added to the lyophilized NALNP as described herein.

[0084] The method for preparing freeze-dried NALNP of the present invention comprises (a) mixing nucleic acid with a lipid mixture solution comprising 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 freeze-drying buffer; and (c) freeze-drying the combined lipid nanoparticles and freeze-drying buffer obtained in (b) to obtain anhydrous freeze-dried NALNP.

[0085] The method for preparing freeze-dried NALNP of the present invention comprises mixing nucleic acid with a lipid mixture solution comprising an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles. The nucleic acid can be any suitable nucleic acid according to the embodiments of the present invention. The lipid mixture solution comprising an ionizable lipid, a structural lipid, a sterol, and a stabilizer can be any suitable lipid mixture according to the embodiments of the present invention. The lipid nanoparticles can be any suitable lipid nanoparticles according to the embodiments of the present invention.

[0086] Any suitable mixing method can be used to form lipid nanoparticles. Lipid nanoparticles according to the embodiments of the present invention can be prepared by standard T-tube mixing techniques, turbulent mixing, titration mixing, agitation-assisted sequential self-assembly, or by passively mixing all components to self-assemble the components into nanoparticles. A variety of methods have been developed to formulate lipid nanoparticles containing gene drugs. For example, suitable methods are disclosed in U.S. Patent Nos. 5,753,613 and 6,734,171. These methods include 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, and obtaining lipid nanoparticles with a nucleic acid encapsulation efficiency of 65-99%. Both methods rely on the presence of an ionizable lipid to achieve nucleic acid encapsulation and on the presence of a stabilizer to inhibit aggregation and the formation of large structures.

[0087] Automated micro-mixing instruments, such as instruments (PrecisionNanoSystems Inc, Vancouver, Canada) are capable of rapidly and controllably producing nanomedicines (liposomes, lipid nanoparticles, and polymer nanoparticles). The instrument achieves controlled molecular self-assembly of nanoparticles through a microfluidic mixing cartridge, which allows for millisecond mixing of nanoparticle components at the nanoliter, microliter, or larger scale, and can be customized or parallelized. Small-scale rapid mixing allows for reproducible control of particle synthesis and quality, which is not possible with large instruments.

[0088] Preferred methods include instruments such as microfluidic mixing devices, for example Spark TM 、Ignite TM 、BenchtopTM and Blaze TM , to achieve encapsulation of nearly 100% of the nucleic acid used during the formation process in the particles in one step. In one embodiment, the lipid particles are prepared by a method that encapsulates about 90% to about 100% of the nucleic acid used during the formation process in the particles.

[0089] U.S. Patent Nos. 9,758,795 and 9,943,846 describe methods using small volume mixing techniques and novel formulations derived therefrom. U.S. Patent No. 10,159,652 describes more advanced methods using small volume mixing techniques and products formulating different materials. U.S. Patent No. 9,943,846 discloses a microfluidic mixer with different paths and holes for the components to be mixed. PCT Publication No. WO2017117647 discloses a microfluidic mixer with a disposable sterile path. U.S. Patent No. 10,076,730 discloses a bifurcated annular micromixing geometry and its application in micromixing. PCT Publication No. WO2018006166 discloses a programmable automated microfluidic mixer and a mixing chip therefrom. Mixing columns with microchannels and mixing geometries for mixing instruments are available from, for example, PrecisionNanoSystems Inc.

[0090] In an embodiment of the present invention, a device for biological microfluidic mixing is used to prepare lipid particles according to an embodiment of the present invention. The device includes first and second reagent flows that are fed into a microfluidic mixer, and the lipid particles are collected from the outlet or enter a sterile environment.

[0091] The first flow includes a therapeutic agent in a first solvent. Suitable first solvents include solvents in which the therapeutic agent is soluble and miscible with the second solvent. Suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers or other low pH buffers.

[0092] The second flow includes a lipid mixture material in a second solvent. Suitable second solvents include solvents in which the ionizable lipid according to an embodiment of the present invention is soluble and miscible with the first solvent. Suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Representative second solvents include 90% aqueous ethanol or absolute ethanol.

[0093] In one embodiment of the present invention, suitable devices include one or more microchannels (i.e., channels with a maximum dimension less than 1 mm). In one example, the diameter of the microchannels is from about 20 to about 300 μm. In an example, at least one region of the microchannel has a main flow direction, and one or more surfaces define therein at least one groove or protrusion having an orientation that forms an angle with the main direction (e.g., a staggered herringbone mixer), as described in U.S. Patent No. 9,943,846, or a bifurcated annular flow, as described in U.S. Patent No. 10,076,730. To achieve the maximum mixing rate, it is advantageous to avoid excessive fluid resistance prior to the mixing region. Thus, one example of the device has a non-microfluidic channel with a dimension greater than 1000 μm for delivering fluid to a single mixing channel.

[0094] Less automated mixing methods and instruments, such as those described in Zhang, S-h et al., Chemical Eng. J. 144(2):324 - 328(2008) and U.S. Published Patent Application US20040262223, and Jeffs, L.B et al., Pharm. Resch., 22(3):362 - 372(2005)), can also be used to produce the lipid particle compositions of the present invention.

[0095] The method for preparing freeze-dried NALNP of the present invention includes combining lipid nanoparticles with a freeze-drying buffer. Any suitable freeze-drying buffer according to the embodiments of the present invention can be used. Any suitable method can be used to combine the lipid nanoparticles with the freeze-drying buffer. For example, in some embodiments, the freeze-drying buffer and the lipid nanoparticles are mixed. In a preferred embodiment, the freeze-drying buffer and the lipid nanoparticles are mixed using a pipette.

[0096] The method for preparing freeze-dried NALNP of the present invention includes freeze-drying the combined lipid nanoparticles and freeze-drying buffer to obtain anhydrous freeze-dried NALNP. In some embodiments, freeze-drying the combined nanoparticles and freeze-drying buffer includes: (a) freezing the combined lipid nanoparticles and freeze-drying buffer at -40 to -90 °C for 60 - 400 minutes, (b) drying the combined lipid nanoparticles and freeze-drying buffer at -20 to -40 °C and 30 - 100 mTorr for 700 - 980 minutes, and (c) drying the combined lipid nanoparticles and freeze-drying buffer at 4 - 10 °C and 30 - 100 mTorr for 250 - 500 minutes.

[0097] Lyophilizing the combined nanoparticles and lyophilization buffer can include freezing the combined lipid nanoparticles and lyophilization buffer. Any suitable method for freezing the lipid nanoparticles and lyophilization buffer can be used. For example, the combined lipid nanoparticles and lyophilization buffer can be directly placed in a refrigerator below zero degrees Celsius, frozen in liquid nitrogen (e.g., immersed for an appropriate time, such as about 30 seconds), or placed in a cryogenic container with controlled freezing and then placed in a refrigerator. The combined nanoparticles and lyophilization buffer can be frozen at any suitable temperature. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are frozen between -40°C and -90°C. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are 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 the range defined by any two of the above values.

[0098] In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are frozen at -60°C or -80°C. The combined nanoparticles and lyophilization buffer can be frozen for any suitable length of time. In some embodiments, the combined nanoparticles and lyophilization buffer are frozen for 60 - 400 minutes. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are 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 for a length of time within the range defined by any two of the foregoing values.

[0099] Lyophilizing the combined nanoparticles and lyophilization buffer can include drying or dehydrating the combined lipid nanoparticles and lyophilization buffer. In some embodiments, the combined nanoparticles and lyophilization buffer are dried once. In some embodiments, the combined nanoparticles and lyophilization buffer are dried more than once. For example, the combined nanoparticles and lyophilization buffer can be dried 1, 2, 3, 4, or 5 times. Any suitable method can be used to dry the combined lipid nanoparticles and lyophilization buffer.

[0100] In some embodiments, the combined lipid nanoparticles and lyophilization buffer are first dried at -20 to -40 °C and 30 - 100 mTorr for 700 - 980 minutes. The combined lipid nanoparticles and lyophilization buffer can be dried at any suitable temperature. In some embodiments, the combined lipid nanoparticles and lyophilization buffer can be dried at a temperature of -20 to -40 °C. For example, the combined lipid nanoparticles and lyophilization buffer can be dried at -20 °C, -22 °C, -24 °C, -26 °C, -28 °C, -30 °C, -32 °C, -34 °C, -36 °C, -38 °C, -40 °C, or at a temperature within the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried at a temperature of -40 °C. The combined lipid nanoparticles and lyophilization buffer can be dried at any suitable pressure. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried at a pressure of 30 - 100 mTorr. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried at 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 at a pressure within the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried at a pressure of 60 mTorr. The combined lipid nanoparticles and lyophilization buffer can be dried for any suitable time. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried for 700 - 980 minutes. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are 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 the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried for 840 minutes.

[0101] In some embodiments, the combined lipid nanoparticles and lyophilization buffer are subjected to a second drying. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are secondarily dried at 4 - 10 °C at 30 - 100 mTorr for 250 - 500 minutes. The combined lipid nanoparticles and lyophilization buffer can be secondarily dried at any suitable temperature. In some embodiments, the combined lipid nanoparticles and lyophilization buffer can be dried at a temperature of 4 to 10 °C. For example, the combined lipid nanoparticles and lyophilization buffer can be dried at 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 at a temperature within the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried at a temperature of 10 °C. The combined lipid nanoparticles and lyophilization buffer can be dried at any suitable pressure. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried at a pressure of 30 - 100 mTorr. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried at 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 at a pressure within the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried at a pressure of 60 mTorr. The combined lipid nanoparticles and lyophilization buffer can be dried for any suitable time. In some embodiments, the combined lipid nanoparticles and lyophilization buffer are dried for 250 - 500 minutes. For example, in some embodiments, the combined lipid nanoparticles and lyophilization buffer are 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 the range of any two of the above values. In a preferred embodiment, the combined lipid nanoparticles and lyophilization buffer are dried for 320 minutes.

[0102] In some embodiments, the method further includes 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 a 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 the range of any two of the above values. In some embodiments, the anhydrous lyophilized NALNP is mixed with the solution at a temperature between 0°C and 10°C. For example, in some embodiments, the anhydrous lyophilized NALNP is mixed 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 the range of any two of the above values. In a preferred embodiment, the anhydrous lyophilized NALNP is mixed with the 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, glucose, saline, or water.

[0103] Embodiment

[0104] 1. A lyophilized nucleic acid lipid nanoparticle (NALNP) comprising

[0105] (a) a lipid nanoparticle comprising a nucleic acid; and

[0106] (b) a lyophilization buffer comprising a sugar and a lyophilization reagent selected from sugar mimetic oligomers / polymers, amphiphilic thermoresponsive polymers, ethylene glycol mimetic polymers, hydrophilic monomers, and hydrophilic polymers.

[0107] 2. The lyophilization buffer according to Embodiment 1, wherein the sugar mimetic oligomer / polymer is octanoyl sucrose, cyclodextrin, β-cyclodextrin polymer, dextran, trehalose, carboxyl-terminated PEG with a sorbitol core, Betadex TM sodium sulfobutyl ether or 2-hydroxypropyl-β-cyclodextrin.

[0108] 3. The lyophilization buffer according to Embodiment 1, wherein the amphiphilic thermoresponsive polymer is poly(N-vinylcaprolactam), poly(N,N-dimethylacrylamide), poly(N,N-diethylacrylamide), or poly(acrylamide).

[0109] 4. The lyophilized buffer according to embodiment 1, wherein the ethylene glycol-mimicking polymer is 6-arm branched PEG, 5-arm branched PEG, 3-arm branched PEG, trimethylolpropane ethoxylate, polyethylene glycol, poloxamer 407, amine-terminated 4-arm PEG, glycerol ethoxylate, or poly(propylene glycol).

[0110] 5. The lyophilized buffer according to embodiment 1, wherein the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglyceryl, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), amino acid, or L-arginine.

[0111] 6. The NALNP according to embodiment 2, wherein the wt / wt ratio of the lyophilization reagent to the sugar is 1:8.

[0112] 7. The NALNP according to embodiment 2, wherein the wt / wt ratio of the lyophilization reagent to the sugar is 1:5.

[0113] 8. The NALNP according to embodiment 1, further comprising a pharmaceutically acceptable diluent selected from the group consisting of solutions of Tris, sodium acetate, sodium citrate, glucose, and saline.

[0114] 9. The NALNP according to embodiments 1-8, wherein the nucleic acid of the NALNP to the lyophilization reagent to the sugar has a wt / wt ratio of 1:125:1000, 1:250:1000, 1:250:2000, or 1:500:2000.

[0115] 10. The NALNP according to embodiment 1, wherein the sugar is sucrose.

[0116] 11. The NALNP according to any one of embodiments 1-8, wherein the NALNP is in an anhydrous form.

[0117] 12. A method for preparing a lyophilized NALNP, comprising

[0118] (a) mixing a nucleic acid with a lipid mixture solution comprising an ionizable lipid, a structural lipid, a sterol, and a stabilizer to form lipid nanoparticles;

[0119] (b) combining the lipid nanoparticles obtained in (a) with a lyophilized buffer comprising a sugar, a lyophilization reagent, and a pharmaceutically acceptable diluent, the lyophilization reagent being selected from polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-β-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 carboxyl-terminated PEG with a sorbitol core; and

[0120] (c) Lyophilize the combined lipid nanoparticles and the lyophilization buffer obtained in (b) to obtain anhydrous lyophilized NALNP.

[0121] 13. The method according to embodiment 12, wherein lyophilizing the combined nanoparticles and the lyophilization buffer comprises: [[ID=%]]

[0122] (a) Freeze the combined lipid nanoparticles and the lyophilization buffer at -40 to -90 °C for 60 - 400 minutes,

[0123] (b) Dry the combined lipid nanoparticles and the lyophilization buffer at -20 to -40 °C and 30 - 100 mTorr for 700 - 980 minutes, and

[0124] (c) Dry the combined lipid nanoparticles and the lyophilization buffer at 4 - 10 °C and 30 - 100 mTorr for 250 - 500 minutes.

[0125] 14. The method according to any one of embodiments 12, wherein the lyophilization buffer comprises a pharmaceutically acceptable diluent.

[0126] 15. The method according to embodiment 14, wherein the pharmaceutically acceptable diluent is Tris, sodium acetate, sodium citrate, glucose, saline or water.

[0127] 16. The method according to any one of embodiments 10 - 15, wherein the wt / wt ratio of the lyophilization reagent of the lyophilization buffer, sugar and pharmaceutically acceptable diluent is between 1:4:40 and 1:8:80.

[0128] 17. The method according to any one of embodiments 10 - 15, wherein the wt / wt ratio of the nucleic acid of the NALNP, lyophilization reagent and sugar is 1:125:1000, 1:250:1000, 1:250:2000 or 1:500:2000.

[0129] 18. The method according to any one of embodiments 10 - 15, wherein the sugar is sucrose.

[0130] The following examples further illustrate the present invention, but should of course not be construed as limiting its scope in any way. [[ID=%]]

[0131] All solvents and reagents are commercial products and used as received, unless otherwise specified. Temperatures are in degrees Celsius. For the examples, the following abbreviations are used:

[0132] hEPO - mRNA: human erythropoietin protein mRNA

[0133] FLuc-mRNA: Firefly luciferase protein mRNA

[0134] eGFP-mRNA: Enhanced green fluorescent protein mRNA

[0135] LB: Lyophilization buffer (lyo buffer)

[0136] saRNA: Self-amplifying mRNA

[0137] eGFP: Basic (constitutive fluorescence) green fluorescent protein derived from Aequorea victoria

[0138] hEPO: Human erythropoietin

[0139] h: Hour

[0140] HPLC: High performance liquid chromatography

[0141] MFI: Median fluorescence intensity

[0142] min: Minute

[0143] mL: Milliliter

[0144] mmol: Millimole

[0145] μL: Microliter

[0146] PBS: Phosphate buffered saline

[0147] wt: Weight

[0148] ℃ or Deg C: Degree Celsius

[0149] IL: Ionizable lipid

[0150] MC3: DLin-MC3-DMA

[0151] Tween 80: Polysorbate 80

[0152] BRIJ TM L4: Polyoxyethylene (4) lauryl ether

[0153] BRIJ TM S10: Polyoxyethylene (10) stearyl ether

[0154] BRIJ TM S20: Polyoxyethylene (20) stearyl ether

[0155] BRIJ TM S35: Polyoxyethylene (23) lauryl ether

[0156] TPGS1000: D-α-Tocopheryl polyethylene glycol 1000 succinate

[0157] VA composition (VA): A lipid mixture containing 47.5 mol% IL, 12.5 mol% DOPE, 38.5 mol% cholesterol, and 1.5 mol% PEG-DMG

[0158] 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

[0159] CT10 composition: A lipid mixture containing 40 mol% ionizable lipid / 20 mol% DSPC / 37.5 mol% Chol / 2.5 mol% BRIJ TM Lipid mixture of S10

[0160] PNI 516: (Z)-3-(2-((1,17-bis(2-octylcyclopropyl)heptadec-9-yl)oxy)-2-oxoethyl)-2-(pent-2-en-1-yl)cyclopentyl 4-(dimethylamino)butyrate (WO 2020 / 252589)

[0161] PNI 127: (2R,3S,4R)-2-(((1,4-dimethylpiperidin-4-carbonyl)oxy)methyl)tetrahydrofuran-3,4-diyl (9E,9'E,12E,12'E)-bis(octadec-9,12-dienoate) (WO 2021 / 000041)

[0162] DLin-MC3-DMA or MC3: Dilinoleyl methyl-4-dimethylaminobutyrate

[0163] DLin-KC2-DMA or KC2: 2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane

[0164] BOCHD-C3-DMA butyrate: 4-(dimethylamino)-,9-(2-octylcyclopropyl)-1-[8-(2-octylcyclopropyl)octyl]nonyl ester

[0165] V101: A cloning vector template for the synthesis of self-amplifying replicon DNA or RNA encoding a target gene (GOI): The parental VEEV TC83 replicon, having a subgenomic promoter and containing a multiple cloning site for inserting any GOI (U.S. Patent No. 7,425,337 to Geall et al.)

[0166] A3, A5: saRNA encoding the SARS-Cov-2 spike protein using the V101 vector (U.S. Patent No. 7,425,337)

[0167] Base Composition Buffer (BC): 10% (w / v) sucrose (10 mg / mL) dissolved in 20 mM Tris buffer and 1X PBS

[0168] Lyo reagent or lyophilized reagent: Components used in the lyophilized buffer (LB) composition

[0169] Lyophilized buffer or Lyo buffer: The combination of lyo reagents (1 - 5% weight / volume, as described in Table 1) in the base composition (BC) buffer is defined as the lyophilized buffer or Lyo buffer (LB).

[0170] Table 1: Reagents and Sources

[0171]

[0172]

[0173] Example 1

[0174] This example confirmed the nucleic acid therapeutic agent (NAT) formulations used in the examples of this application. Messenger RNA, saRNA, or DNA plasmid nucleic acid therapeutic agent (NAT) as described below was diluted to the desired concentration using sodium acetate buffer. The RNA / pDNA was dissolved in 100 mM sodium acetate buffer to reach the desired concentration of ~168 μg / mL. The concentration of NAT was determined by Nanodrop (Thermo Scientific TM ) and this information was used to determine the required concentration of NAT to be mixed with the lipid mixture in ethanol in the Ignite TM instrument as described in Example 2.

[0175] Example 2

[0176] This example confirmed the preparation of lipid nanoparticles (LNP) by microfluidic mixing used in the examples of this application. NAT was prepared as shown in Example 1. The components of the lipid mixture included ionizable lipids, structural lipids or helper lipids, sterols, and stabilizers in different molar ratios. Stabilizers refer to any reagent including PEG - DMG, or as defined in the description above under the category of stabilizing LNP formation. The lipid mixture composition was prepared by combining specified amounts of lipid stock solutions from ethanol (12.5, 25, or 37.5 mM as needed; a mixture of ionizable lipid, structural lipid, sterol, and stabilizer, as described in Table 2) in ethanol. Then, the lipid mixture composition and NAT were run through the Ignite TM microfluidic mixer to prepare LNP.

[0177] The mixing of nucleic acid therapeutics (NAT) and lipids is carried out as follows. Ionizable lipids, helper lipids, sterols, and stabilizers are 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). An aqueous phase is prepared by diluting the nucleic acid therapeutic (NAT), such as an mRNA / saRNA / pDNA solution, in 100 mM sodium acetate buffer (pH 4). Unless otherwise specified, use Ignite TM With Ignite TM NxGen TM (DVBM) cartridge (PrecisionNanosystems) to combine the solutions at a flow rate ratio of 1:3 (organic phase: aqueous phase) with an N / P ratio of 8 or 10 and a total flow rate of 12 mL / min. The resulting LNP is diluted 25 - 40 times in 1X PBS (pH 7.4), and the mixture is subjected to downstream processing. Downstream processing includes removing ethanol by dialysis in PBS (pH 7) or using an Amicon TM centrifugal filter (Millipore, USA) or using a tangential flow filtration system. The particles are concentrated to the desired target dose.

[0178] Table 2: Exemplary lipid mixture compositions defined by the ionizable lipid / structural lipid / sterol / stabilizer ratios.

[0179] The stabilizer can be replaced with PEG - DMG.

[0180]

[0181] Example 3

[0182] This example describes the methods for measuring the size, polydispersity index (PDI), and encapsulation efficiency (EE) of LNP in the following examples.

[0183] Use a ZetaSizer TM Nano ZS TM (Malvern Instruments) to measure the size and PDI of LNP by dynamic light scattering (DLS). A He / Ne laser with a wavelength of 633 nm is used as the light source. The data is measured from the scattered intensity data in the backscattering detection mode (measurement angle = 173°). 0.5 to 2 μL of the sample is placed in a cuvette and diluted with PBS (0.3 mL). The measured values are the average of 10 runs for two cycles of each sample. The Z - average size is reported as the particle size and is defined as the harmonic intensity - weighted average particle diameter. By Quant - iT TM RNA reagents were used to measure the EE of the LNPs. These LNP characteristics and the nucleic acid EE results of the LNPs in various lyophilization buffers (LB) are described in the following examples.

[0184] Example 4

[0185] This example describes the lyophilization process and how the lyophilization buffers were evaluated in the following examples.

[0186] Lyophilization buffers (lyo buffers) were prepared in a base composition (BC, 10% sucrose in 20 mM Tris and 1X PBS). The weight / volume percentages of each lyo reagent in the base composition are described in Table 3. As described in Example 2, once the LNPs reached the desired target concentrations (40, 80, and 120 μg / mL) after manufacture, they were mixed with the lyophilization buffer at ratios of 1:1, 1:2, and 1:4 (V:V). The final solution volume of 200 to 300 μL was transferred to UPLC 2 mL glass vials or Afton's Ready-To- Sterile vials (2 mL). Once the desired concentration was reached, the LNP mixed with the lyo buffer was filter-sterilized using a 0.2 μm filter under sterile conditions.

[0187] The LNP mixed with the lyo buffer was lyophilized using a lyophilizer (freeze dryer) instrument (SP Scientific model ADP-S2XL-E0A-X; serial number 326328) by freezing at -60 °C for 3 hours, then first dehydration at -40 °C / 0 min / 840 min / 60 mTorr, and then second dehydration at 10 °C / 0 min / 320 min / 60 mTorr. Figure 1 A representative schematic diagram of the lyophilization process workflow is shown, which details the different drying cycles.

[0188] After secondary drying was completed, the obtained lyophilized cake or lyo cake was stored at different temperatures (RT, 4 °C, or -20 °C) for 24 hours, 1 week, 3 months, or 6 months (the following examples). After the required storage time, unless otherwise specified, the lyo cake was reconstituted to the target NAT concentration / volume (1:1, V / V) in 1X PBS before any cell or animal treatment as described in the following examples. After reconstitution, the final encapsulation efficiency was measured using the Quant-iT TM RNA Reagents and Kits (Invitrogen), and the size of the LNPs was measured using a ZetaSizer TM Nano ZS TM (Malvern Instruments) by dynamic light scattering (DLS).

[0189] To evaluate the efficiency of the lyo buffer, different mRNAs (eGFP mRNA, hEPO mRNA, or FLuc mRNA), saRNAs (A3 or A5), or pDNAs were used to prepare LNP formulations in 47.5 mol% IL / 12.5 mol% DOPE / 38.5 mol% Chol / 1.5 mol% PEG-DMG (VA), 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG (VB), or 40 mol% ionizable lipid / 20 mol% DSPC / 37.5 mol% cholesterol / 2.5 mol% BRIJ TM S10 (CT10) compositions to prepare LNP formulations as described in the following examples. The efficacy of the lyo buffer was evaluated using LNPs encoded with hEPO mRNA or FLuc mRNA containing various ionizable lipids and co-lipids. The lyophilized compositions were compared to the base composition ("BC", 10% sucrose in 20 mM Tris and 1X PBS, in solution form or when reconstituted) as lyophilized LNP cakes or, when possible, as frozen suspensions (BC, -80 °C), or to fresh LNPs (not lyophilized) in PBS.

[0190] Table 3: Lyophilization buffer (LB) compositions

[0191]

[0192]

[0193] **Use 10 mM Tris buffer instead of 20 mM.

[0194] Example 5

[0195] To find a successful mRNA vaccine lyophilization protocol, various lyo buffer compositions were evaluated for the delivery of saRNAs encoding vaccine antigens. SaRNAs encoding the full-length spike protein were used. The physicochemical properties of the lyophilized LNPs (after lyophilization), such as size, polydispersity (PDI), and encapsulation efficiency (EE), were measured and compared to the BC control (LNPs lyophilized with BC) and fresh LNPs (without lyophilization).

[0196] LNPs encapsulating saRNA encoding spike antigens (SARS-Cov-2 spike protein, A3b, A3p, or A5) with PNI 516 as the ionizable lipid were prepared at target concentrations of 40, 80, and / or 120 μg / mL and diluted to a solution volume of 200 μL in 1:1, 1:2, and 1:4 LNP to lyo buffer (v:v) in 2 mL glass vials of UPLC. The LNP formulation compositions and lyo buffer compositions are described below. The LNPs were lyophilized with different lyo buffers added to the base composition of 10% sucrose and 20 mM Tri. Dehydration was carried out by freezing at -60 °C for 3 hours, then dehydrating at -40 °C / 0 min / 840 min / 60 mTorr, and then dehydrating a second time at 10 °C / 0 min / 320 min / 60 mTorr. The lyophilized cakes were stored at RT for 24 hours. For LNPs containing A3 saRNA, additional tests were performed on lyo cakes stored at -20 °C, 4 °C, and RT for one week. Reconstitution was carried out in 200 μL PBS.

[0197] After reconstitution in 1X PBS, size, PDI, and encapsulation efficiency were measured. LB#107, 163, 186, 189, and 192 showed good performance in protecting the size and EE of PNI 516 LNPs encapsulating A3 saRNA within the therapeutically viable range (Table 4). LB#107, 110, 164, 168, 178, 179, 181, 182, 186, and 189 showed good performance in protecting the size and EE of LNPs with PNI 516 and A5 saRNA, where the lyo cakes were stored at RT for 24 hours (Table 5). Figure 2 and Figure 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 the LNPs within the therapeutically relevant range. LB#110 was found to have the smallest size variation, independent of the payload tested. LB#189 retained its minimum PDI, as shown in Table 4.

[0198] For some LBs in Table 1, such as sucrose alone, they were not further pursued due to limitations in early results. For example, LB#3 (sucrose, 25%) was of high viscosity. Additionally, based on the data we obtained, no improvement in CQA and potency was found when the sucrose percentage was increased from 10% to 25% without additional lyophilization reagents. 10% sucrose in 20 mM Tris buffer was established as the base composition (BC) for adding lyophilization reagents and used as a control in the experiments.

[0199] Table 4: Physicochemical properties of freeze-dried and reconstituted spike protein-encoding A3 saRNA LNPs using ionizable lipid 516 and lipid mixture 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG

[0200]

[0201] Table 5: Physicochemical properties of LNPs containing IL PNI 516 and A5 saRNA before and after freeze-drying using the same lipid mixture

[0202]

[0203]

[0204] Example 6

[0205] This example confirmed the potency of freeze-dried saRNA LNPs using Western blot. According to the conventional formulation procedure mentioned in the above examples, LNPs containing PNI 516 encoding the full-length spike protein of SARS-CoV-2 were formulated with a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG composition. Various lyo buffer compositions were tested to maintain the in vitro potency of saRNA LNPs. The freeze-dried cakes were then reconstituted with 1X PBS, sterilized, analyzed (size and EE) and used for in vitro cell treatment. LNPs freeze-dried with BC composition (BC) and PBS treatment (without LNPs) were used as controls.

[0206] Western blot: HEK-293 cells were seeded in 6-well plates (0.3 x 10 6 cells / well) for 2 days and then treated with 1 μg / mL of the freeze-dried sample for 1 day. Cell lysates were prepared using IP lysis buffer. Protein quantification was performed using the BCA kit according to the supplier (Pierce TM BCA Protein Assay Kit) protocol. Protein samples were denatured using loading dye and 2-mercaptoethanol. SDS-PAGE was run in a mini gel electrophoresis tank (175 V, 500 mA, 90 minutes). The gel was transferred to a nitrocellulose membrane (Thermo Fisher) using the iBlotTM2 device (Thermo Fisher). After blocking the membrane, the membrane was stained with the first antibody on a shaker at RT for 2 h and then with the second antibody. 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 compared with the control to evaluate the performance of the freeze-dried LNPs.

[0207] Figure 4 is a Western blot of the cellular proteins of the treated cells. Compared with the BC lyo-cakes, the LNPs lyophilized with LB#107, 110, 163, 164, 180, 186, and 189 showed the same or superior performance. In this case, the lyo-cakes were stored at RT for 24 hours. In another experiment, the lyophilized A3 saRNA LNP cakes were stored for one week at three different temperatures (-20 °C, 4 °C, and RT). As Figure 5 and Figure 6 shown, LB#163 showed superior spike protein expression performance at all three different temperatures. Figure 5 and Figure 6 also showed that LB#107, 186, and 192 retained saRNA activity at -20 °C and 4 °C. To evaluate the long-term in vitro potency of LB#163, the A3 saRNA LNP lyo-cakes containing LB#163 were stored at 4 °C and RT for three months. Compared with BC, LB#163 protected saRNA activity at both temperatures, especially at 4 °C. The results are as Figure 7 shown.

[0208] Example 7

[0209] This example confirmed the ability of the lyophilization buffer to maintain the activity of the saRNA-LNP vaccine in mice. Mice were given (IM) the lyophilized LNP, which used the VB composition containing the ionizable lipid PNI 516 and encoded the SARS-COV-2 full-length spike protein (A5 saRNA), reconstituted in 1X PBS. The lyophilized vaccine (cake) was stored at -20 °C, 4 °C, and RT for 7 days. PBS and cryopreserved LNP with BC were used as controls. Blood was drawn from the tail vein 42 days later, and serum was prepared according to standard procedures. A 6-week serum analysis of SARS-CoV-2 spike protein-specific IgG was performed using an established ELISA procedure.

[0210] Nunc Maxisorp was coated with 0.5 μg / ml of SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Cat. No. 40589-V08B1, Sino Biological, Beijing, China) using PBS TMThe ELISA plate was coated overnight. Mouse serum was diluted (1:40000) using 1X ELISA Assay Diluent B (5X) (Cat. No. 421205, BioLegend, San Diego, USA). Standards (10 - 0.16 ng / ml) were prepared using SARS-CoV-2 (2019-nCoV) Spike Neutralizing Antibody Mouse Mab (Cat. No. 40591-MM43, Sino Biological). HRP Goat anti-Mouse IgG (minimal cross-reactivity) antibody (Cat. No. 405306, BioLegend) was used for detection. Western TMB Substrate was used for color development, terminated with HCl, and readings were recorded at 450 nm.

[0211] Figure 8 It is the measurement of SARS-CoV Spike protein-specific IgG. At all three different storage temperatures (-20 °C, 4 °C, and RT), LB#163 performed equally well compared to the frozen-stored LNP control.

[0212] Example 8

[0213] This example confirmed the ability of the lyophilization buffer of the present invention to protect mRNA LNPs encoding human EPO protein (hEPO).

[0214] Culture conditions for HEK 293 cells for EPO expression: HEK 293 cells were seeded at 0.3 x 10 6 cells / well in a 6-well plate in 2 mL of complete DMEM (Gibco) and allowed to grow at 37 °C in 5% CO2 for 48 hours. The lyophilized hEPO mRNA-LNP was stored at the desired temperature for 24 hours, reconstituted in 1X PBS, and added to the cells at 1 μg / mL mRNA along with the control. After incubation for 48 hours, the cell suspension was harvested and centrifuged at 1200 g for 5 minutes. The supernatant was analyzed on an ELLA TM instrument (Protein Simple by Biotechne TM ) using the Simple Plex TM Human Erythropoietin Cartridge to measure the concentration of hEPO protein.

[0215] Lyophilization: LNPs encoding hEPO mRNA were formulated as described in the above examples. Four leading lyo buffer compositions were tested to maintain the in vitro potency of the mRNA LNPs, which were formulated with the VB composition (47.5 mol% PNI 516 / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG). LNPs lyophilized with BC or treated with PBS (without LNPs) were used as controls. The 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 as Figure 9 shown. Fresh LNPs had better activity than LNPs lyophilized with BC buffer, indicating that the basal composition containing 20 mM Tri and 10% sucrose could not protect the mRNA when the LNPs were lyophilized. LNPs lyophilized and reconstituted with lyo buffers (LB#163, #189, #110, and #107) maintained their mRNA activity at both temperatures (4 °C and RT) and were more effective than the BC buffer composition. The corresponding sizes and encapsulation efficiencies (%) of the lyophilized LNPs were measured and are shown in Table 6.

[0216] Table 6: Physicochemical properties of lyophilized hEPO mRNA-containing LNPs containing ILPNI 516 using the lipid mixture VB composition compared to control LNPs

[0217]

[0218] Example 9

[0219] This example demonstrated the ability of the lyophilization buffer of the present invention to protect mRNA LNPs encoding human EPO protein (hEPO).

[0220] In another study using hEPO mRNA, the encapsulation efficiency (%) and size data of the reconstituted (1X PBS) lyo cakes of PNI 516 and hEPO LNPs were measured after 7 days of storage of the lyo cakes at the designated temperatures (-20 °C, 4 °C, or RT). LNP formation and lyophilization were as described in Example 8. The results are shown in Table 7. Fresh LNPs and LNPs lyophilized in BC were shown as positive controls at the corresponding temperatures (-20 °C, 4 °C, or RT). LB#107, 110, 163, 186, and 189 retained the size and EE of the LNPs within the therapeutic range (Table 7).

[0221] Table 7: Physicochemical properties of lyophilized hEPO mRNA-containing LNPs containing IL PNI 516 using the lipid mixture VB composition after 7 days of storage at different temperatures

[0222]

[0223]

[0224] Example 10

[0225] This example demonstrated the efficacy of lyo buffers using different lipid mixture compositions by varying the ionizable lipid (IL) or 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 the mRNA. The lyophilized LNPs were stored under appropriate test conditions and / or time periods and the RNA integrity was tested in HEK-293 cells using an in vitro potency assay.

[0226] Lyophilization. 100 μL of 80 μg / mL LNPs encapsulating hEPO mRNA were mixed with 100 μL of buffer and lyophilized. After lyophilization, the cakes were stored at RT for 7 days. The cakes were reconstituted in 1X PBS and the size and encapsulation efficiency (%) were determined. LNPs stored at -80 °C using BC were used as the positive control.

[0227] hEPO mRNA-LNPs were encapsulated in a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG composition using different ionizable lipids PNI 516, PNI 127, MC3, KC2, and BOCHD-C3-DMA, as well as controls such as fresh LNPs (without lyophilization) and BC lyophilized LNPs. The lyo cakes were stored at RT for 7 days and reconstituted in 1X PBS. The results are shown in Figure 10 where lyo buffers LB#189, 110, and 107 retained the expression of EPO better than BC and fresh LNPs. The corresponding size and encapsulation efficiency (%) were measured and are shown in Figure 11 and Figure 12 .

[0228] hEPO mRNA-LNPs were prepared at a molar ratio of PNI516:Chol:helper lipid:PEG-DMG (47.5:38.5:12.5:1.5) by varying the helper lipid DSPC, DOPE, DPPC, or DOPC and lyophilized. The lyophilized cakes were stored at RT for 7 days, reconstituted with 1X PBS, and analyzed for size and encapsulation efficiency (%) (13). The expression of erythropoietin in HEK293 cells was measured using a Simple Plex TM HumanErythropoietin Cartridge on an ELLA TM instrument ( Figure 13 ). LB#107 protected the activity of the LNPs regardless of the helper lipid used in the LNP composition (Figure 13 A For LNPs made of DSPC, DPPC, DOPE, DOPC, LB#107; Figure 13 B For LNPs made of DSPC, LB#107 and LB#110; Figure 13 C For LNPs made of DPPC, LB#107 and 189).

[0229] After storing the reconstructed cakes for 7 days at the specified temperature, the corresponding encapsulation efficiency (%) of the reconstructed cakes is shown in Table 8. Fresh LNPs (not lyophilized) and the lyophilized cakes of the base composition (BC) at the corresponding temperatures are shown as positive controls.

[0230] Table 8: Physicochemical properties of lyophilized hEPO mRNA-containing LNPs with IL PNI516 using lipid mixtures with different helper lipids after storing for 7 days at room temperature (RT)

[0231]

[0232] Example 11

[0233] This example demonstrated the efficacy of the lyophilization buffer in protecting mRNA-LNP encoding the luciferase protein.

[0234] Firefly luciferase protein expression in HEK cells: HEK 293 cells (ATCC) were seeded in white 96-well plates at a density of 12×10 3 cells / well in 100 μL of complete DMEM (Gibco) and allowed to grow for 24 hours at 37 °C in 5% CO2. The lyophilized mRNA LNP encoding the firefly luciferase protein was reconstituted in 1X PBS and added to the cells at doses in the range of 50 ng along with other controls. After 24 hours of treatment, the cell viability of the cells was analyzed, and luciferase expression was measured using the ONE-Glo TM Tox Assay Kit (Promega) according to the manufacturer's protocol.

[0235] Lyophilization: Fluc mRNA-LNP was prepared in the VB composition with PNI 516 and 127 at N / P -8 and lyophilized by mixing with LB#107. The lyo cakes were stored at RT for one week and reconstituted in 1X PBS. The control LNP was BC-treated LNP stored at -80 °C (BC, -80 °C). The results are as Figure 14As shown, compared with the control BC stored at -80°C, LB#107 showed similar or better luciferase protein expression activity in HEK cells with two lipids (PNI516 and 127). Table 9 shows that LB#107 showed good performance in terms of the size and EE of the LNP lyophilized cakes of PNI 516 and 127 encoding luciferase protein mRNA stored at RT for one week.

[0236] Table 9: Physicochemical properties of lyophilized LNP containing luciferase protein-encoding mRNA with a lipid mixture of 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG composition compared to control LNP

[0237]

[0238] **LNP was stored in BC (10% sucrose, 10 mM Tris) at -80°C and measured after thawing.

[0239] Example 12

[0240] This example confirmed that the lyophilization buffer of the present invention retained the activity of mRNA-LNP in mice: EPO expression in C57BL / 6 mice. Reconstituted lyophilized LNP (cakes stored at -20°C, 4°C, and RT for 7 days) and fresh LNP containing EPO-encoding mRNA were administered to mice. All formulations were made with PNI 516, which contained a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG composition. Blood was drawn from the tail vein, and serum was prepared according to standard procedures. Using the ELLA Simple Plex TM Human Erythropoietin Cartridge kit (ProteinSimple of Biotechne TM ), a 6-hour serum assay for EPO expression in mouse serum was evaluated by automated ELISA.

[0241] The EPO levels in mice were as Figure 15 shown. LB#107 was the best-performing test candidate at RT and 4°C, but LB#189 was the best at -20°C. Freshly prepared LNP stored in PBS at 4°C was used as a control. Compared with the fresh LNP control, all reconstituted lyophilized LNP showed similar or better potency regardless of the 7-day aging at various storage temperatures. Encapsulation efficiency was as Figure 16As shown, LB#107, 110, and 189 retained EE after lyophilization. The sizes and PDI of these same LNPs are as Figure 17 shown. Lyophilization had no substantial effect on size and PDI compared to the control.

[0242] Example 13

[0243] This example demonstrated that the lyophilization buffer of the present invention protects plasmid-encapsulated LNPs. The CMV-eGFP-pDNA plasmid custom-made by GenScript USA Inc, Piscataway, NJ and PNI 516 lipids were used for the LNP formulation. The LNPs were prepared as described above. The plasmid was formulated with PNI 516, which contains a 47.5 mol% IL / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG composition, with an N / P ratio of 8. Then the LNP samples were lyophilized, and the lyo cakes were stored at RT for one week. After reconstitution in 1X PBS, the size and EE were measured. As shown in Table 10, LB#107, 110, and 189 protected the size and PDI of the LNPs. The average diameter of all tested LNPs was 93 - 115 nm, and the PDI was 0.2, and the encapsulation efficiency before and after lyophilization and reconstitution was greater than 80%.

[0244] Table 10: Physicochemical properties of lyophilized eGFP pDNA-containing LNPs compared to control LNPs

[0245]

[0246] Example 14

[0247] This example demonstrated that the lyophilization buffer of the present invention protects eGFP mRNA. As described in Example 2, LNPs were prepared using PNI516 and the lipid composition VA. As described in Example 5, the LNPs were lyophilized using lyophilization buffers (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, and then the lyophilized LNPs were measured. Each lyo buffer protected the size and EE of the PNI 516 LNPs encoding eGFP mRNA. The lyo buffer LNPs showed similar average size, PDI, and EE to the fresh LNPs (Table 7b).

[0248] Table 11: Physicochemical properties of GFP mRNA-containing LNPs using lipid mixture composition VA before and after lyophilization

[0249]

[0250] Example 15

[0251] This example demonstrated how the selected lyophilized reagents protected the LNPs. As described above, LNPs containing 51.647 mol% PNI / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG were prepared, which encapsulated mRNA encoding the full-length spike protein of SARS-CoV-2. The ability of lyo buffers #101, 109, 112, 113, 119, 120, 134, 135, 136, 140, 142, 143, 147, 148, 157, 161, 162, 166, 171, 182, and 187 (as described in Table 3 above) to maintain the in vitro potency of the saRNA LNPs was tested. The lyophilized cakes were then reconstituted with 1X PBS, sterilized, analyzed, and used for cell treatment.

[0252] Western blot: HEK-293 cells were seeded in 6-well plates (0.3 x 10 6 cells / well) for 2 days and then treated with 1 μg / mL of the lyophilized samples for 1 day. Cell lysates were prepared using IP lysis buffer. Protein quantification was performed using the BCA kit according to the supplier's (Pierce TM BCA Protein Assay Kit) protocol. Protein samples were denatured using loading dye and 2-mercaptoethanol. SDS-PAGE was run in a mini gel electrophoresis tank (175 V, 500 mA, 90 minutes). The gel was transferred to a nitrocellulose membrane (Thermo Fisher) using the iBlot TM 2 device (Thermo Fisher). After blocking the membrane, the membrane was stained with the first antibody at RT on a shaker for 2 hours and then with the second antibody. 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 compared with the control to evaluate the performance of the lyophilized LNPs. Figure 18 The Western blot results of protein expression in cells treated with NALNPs lyophilized with 31 LB candidates are shown. Protein samples from LNPs lyophilized with LB #54, 120, 134, 135, 148, 157, 167, 168, 187, and 208 showed significant activity after lyophilization.

[0253] Example 16

[0254] Table 12: Encapsulation efficiency (EE) of LNPs encapsulating A3 saRNA with the composition of PNI 516 47.5 mol% / 12.5 mol% DSPC / 38.5 mol% Chol / 1.5 mol% PEG-DMG after 12 days of storage of cakes at RT. Compared with LB#54, LNPs lyophilized with LB#120, 163, 166 and 167 resulted in higher EE%.

[0255] Table 12: Encapsulation efficiency after 12 days

[0256] LB# Reconstructed EE (%) 54 56.2 120 78.3 163 77.3 166 83.5 167 60.5

[0257] Example 17

[0258] Compared with LB#54, the leading LB#108, LB#167, LB#218 and LB#241 containing mRNA LNPs showed a size of less than 100 nm, which is a quality attribute required for parenteral injection. In Table 13, after 2 months of storage of cakes at 4 °C, the size of LNPs containing ILPNI 516 and hEPO mRNA with the composition of 40 mol% iL / 12.5 mol% DSPC / 46.0 mol% cholesterol / 1.5 mol% PEG-DMG showed a great advantage over LB#54 (i.e., PVA).

[0259] Table 13: hEPO mRNA LNP size

[0260] LB# Reconstructed Size (nm) 54 130 108 78 167 82 218 69 241 74 260 73

[0261] Example 18

[0262] The size change of LNPs containing IL PNI 516 and A5 saRNA using the above composition after 1 month of storage of cakes at 4 °C was studied. The leading LB#108, LB#162, LB#164 and LB#167 containing A5 saRNA LNPs showed a size of less than 100 nm, which is a quality attribute required for parenteral injection.

[0263] Table 14: Size of A5 saRNA LNPs after 1 month of storage at 4 °C

[0264] LB# Reconstructed Size (nm) 54 150 108 108 162 89 164 94 167 97

[0265] Example 19

[0266] In vivo EPO expression

[0267] This example describes the procedure for in vivo evaluation of hEPO protein expression of hEPO mRNA-LNP (reconstituted after 2 months of storage in cake form at 4°C). The LNP was 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 at 6 hours and 24 hours after injection. For serum preparation, after collecting whole blood, the collection tube was placed at room temperature for 15 - 30 minutes to allow the blood to clot. The clot was removed by centrifuging the tube at 1000 - 2000 x g for 10 minutes at 4°C. The clear golden-yellow supernatant was carefully removed and transferred to a sterile screw-cap clear polypropylene tube on ice. Then the serum was stored at -80°C until further use. The SARS-CoV-2 antigen-specific IgG level in the serum was measured using an enzyme-linked immunosorbent assay (ELISA). Leads LB#108, LB#164, and LB#167 showed better activity than LB#54. Compared with PVA containing LB#54, leads LB#108, LB#167, LB#218, LB#241, and LB#260 retained activity( Figure 19 ).

[0268] Example 20

[0269] In vivo vaccine expression

[0270] This example describes the procedure for in vivo evaluation of SARS-CoV-2 expression of SARS-CoV-2-expressing A5PNI saRNA-LNP reconstituted after 1 month of storage in cake form at 4°C. On day 0, the LNP was 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, the blood was allowed to clot at RT for 15 - 30 minutes after collection. The clot was removed by centrifuging the tube at 1000 - 2000 x g for 10 minutes at 4°C. The clear golden-yellow supernatant was carefully removed and transferred to a sterile screw-cap clear polypropylene tube on ice. Then the serum was stored at -80°C until further use. The SARS-CoV-2 antigen-specific IgG level in the serum was measured using an enzyme-linked immunosorbent assay (ELISA). As Figure 20 shown, leads LB#108, LB#164, and LB#167 showed better activity than LB#54.

[0271] In the context of describing the present invention (especially in the context of the appended claims), the use of the terms "a", "an", "the", "at least one", and similar references should be construed to cover both the singular and the plural, unless otherwise specified herein or the context clearly dictates otherwise. The use of the term "at least one" after a list of one or more items (e.g., "at least one of A and B") should be construed to mean either one (A or B) selected from the listed items, or any combination of two or more of the listed items (A and B), unless otherwise specified herein or the context clearly dictates otherwise. Unless otherwise specified, the terms "comprising", "having", "including", and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to"). Unless otherwise specified herein, the recitation of a range of values herein is merely intended as a shorthand method of referring separately to each individual value within the range, and each individual value is incorporated into the specification as if it were recited herein individually. All methods described herein can be performed in any suitable order, unless otherwise specified herein or the context clearly dictates otherwise. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illuminate the invention and does not pose a limitation on the scope of the invention, unless otherwise required. Any language in the specification should not be construed as indicating any non-claimed element as essential to the practice of the invention.

[0272] Preferred embodiments of the present invention are described herein, including the best mode known to the inventors for practicing the present invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the above description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend for the present invention to be practiced in other applications than those specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, the present invention includes any combination of all possible variations of the above-described elements, unless otherwise specified herein or the context clearly dictates otherwise.

[0273] All references cited herein, including publications, patent applications, and patents, are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

Claims

1. A lyophilized nucleic acid lipid nanoparticle (NALNP), which comprises (a) a lipid nanoparticle, which lipid nanoparticle comprises nucleic acid; and (b) a lyophilization buffer, which lyophilization buffer comprises a sugar and a lyophilization reagent selected from sugar-mimicking oligomers / polymers, amphiphilic thermoresponsive polymers, ethylene glycol-mimicking polymers, hydrophilic monomers and hydrophilic polymers.

2. The lyophilized buffer according to claim 1, wherein the sugar mimetic oligomer / polymer is n-octanoyl sucrose, cyclodextrin, β-cyclodextrin polymer, dextran, trehalose, carboxyl-terminated PEG with a sorbitol core, Betadex TM sodium sulfobutyl ether or 2-hydroxypropyl-β-cyclodextrin.

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

4. The lyophilization buffer according to claim 1, wherein the ethylene glycol-mimicking polymer is 6-arm branched PEG, 5-arm branched PEG, 3-arm branched PEG, trimethylolpropane ethoxylate, polyethylene glycol, poloxamer 407, amine-terminated 4-arm PEG, glycerol ethoxylate or poly(propylene glycol).

5. The lyophilization buffer according to claim 1, wherein the hydrophilic monomer or polymer is propylene glycol, glycerol, polypropylene glycol, triglyceryl, poly(vinylpyrrolidone), poly(2-ethyl-2-oxazoline), amino acids or L-arginine.

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

8.

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

5.

8. The NALNP according to claim 1, further comprising a pharmaceutically acceptable diluent selected from solutions of Tris, sodium acetate, sodium citrate, glucose and saline.

9. The NALNP according to claim 1, 6 or 7, wherein the wt / wt ratio of the nucleic acid of the NALNP to the lyophilization reagent to the sugar is 1:125:1000, 1:250:1000, 1:250:2000 or 1:500:2000.

10. The NALNP according to claim 1, wherein the sugar is sucrose.

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

12. A method for preparing a lyophilized NALNP, comprising (a) mixing nucleic acid with a lipid mixture solution comprising an ionizable lipid, a structural lipid, a sterol and a stabilizer to form a lipid nanoparticle; (b) combining the lipid nanoparticle obtained in (a) with a lyophilization buffer, which lyophilization buffer comprises a sugar, a lyophilization reagent and a pharmaceutically acceptable diluent, the lyophilization reagent being selected from polyvinylpyrrolidone, poly(N-vinylcaprolactam), 2-hydroxypropyl-β-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 carboxyl-terminated PEG with a sorbitol core; and (c) lyophilizing the combined lipid nanoparticle and lyophilization buffer obtained in (b) to obtain an anhydrous lyophilized NALNP.

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

14. The method according to any one of claims 12, wherein the lyophilization buffer comprises a pharmaceutically acceptable diluent.

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

16. The method according to any one of claims 10 - 15, wherein the wt / wt ratio of the lyophilization reagent of the lyophilization buffer, sugar and pharmaceutically acceptable diluent is between 1:4:40 and 1:8:

80.

17. The method according to any one of claims 10 - 15, wherein the wt / wt ratio of the nucleic acid of the NALNP, lyophilization reagent and sugar is 1:125:1000, 1:250:1000, 1:250:2000 or 1:500:2000.

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

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