Lyophilized and frozen MRNA-LNP formulations

Abstract

The present invention relates to lyophilized and frozen pharmaceutical formulations comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant or cryoprotectant and a buffer, and wherein the lyoprotectant or cryoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG). Methods of making the lyophilized and frozen formulations are also provided.

Description

LYOPHILIZED AND FROZEN MRNA-LNP FORMULATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority benefit of U.S. Provisional Patent Application Serial. No. 63 / 665,199, filed on June 27, 2024, the entire contents of which are incorporated herein by reference for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to lyophilized or frozen pharmaceutical formulations comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant or cryoprotectant and a buffer, and wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG). Methods of making the lyophilized or frozen formulations are also provided.BACKGROUND OF THE INVENTION

[0003] The typical requirement for ultra-cold storage poses significant logistical challenges for global distribution of therapeutics. Lyophilization serves as a potential strategy to extend nucleic acid (NA)-lipid nanoparticle (LNP) (NA-LNP) shelf life while eliminating the need for ultra-cold logistics. While recent advancements have demonstrated the potential of lyophilization, there remains a gap in comprehensive comparisons of lyoprotectants and buffers. The lyophilization of lipid nanoparticles is also a nuanced process, where even subtle parameter changes can influence performance. Throughout the lyophilization process, NA- LNPs face various stressors, including but not limited to crystallization, alterations in ionic strength, interfacial stress between ice and liquid, and more. The stresses induced during lyophilization may introduce variations in formulation quality attributes, such as particle size, cargo encapsulation, NA integrity, as well as LNP structures and in vitro activity. Therefore, there is an emerging need to broaden the selection of lyoprotectants and buffers for the lyophilization development, and therefore comprehend their influence on critical quality attributes of NA-LNPs.BRIEF SUMMARY OF INVENTION

[0004] The present invention provides a lyophilized or frozen pharmaceutical formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant or cryoprotectant and a buffer, and wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG). The present invention further provides a method of making the lyophilized or frozen formulations.

[0005] In one aspect of the invention, there is provided a lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant and a buffer; wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG).

[0006] In some embodiments according to any of the embodiments described above, the PVP is PVP-K12, PVP-K15, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

[0007] In some embodiments according to any of the methods described above, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

[0008] In some embodiments according to any of the embodiments described above, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0009] In some embodiments according to any of the embodiments described above, the nucleic acid comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA is a circular DNA or a linear DNA. In some embodiments, the DNA is a plasmid DNA. In some embodiments, the DNA is a genomic DNA. In some embodiments, the DNA is a synthetic DNA. In some embodiments, the synthetic DNA is a cDNA.

[0010] In some embodiments, the nucleic acid comprises a ribonucleic acid (RNA). In some embodiments, the RNA is a circular RNA or a linear RNA. In some embodiments, the RNA is messenger RNA. In some embodiments, the RNA is a micro RNA (miRNA). In some embodiments, the RNA is a small inhibitory RNA (siRNA). In some embodiments, the RNA is a guide RNA (gRNA).

[0011] In some embodiments, the DNA or RNA is an antisense oligonucleotide.

[0012] In some embodiments according to any of the embodiments described above, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k.

[0013] In some embodiments according to any of the embodiments described above, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v). In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the lyoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 20% or 30% weight per volume (w / v). In some embodiments, the lyoprotectant is is 5% PVP-K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25. In some embodiments, the lyoprotectant is PEG, wherein the lyoprotectant is 10% PEG- 1500.

[0014] In some embodiments according to any of the embodiments described above, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or the buffer is a phosphate buffer. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

[0015] In some embodiments according to any of the embodiments described above, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is between pH 4.0 and pH 8.0. In someembodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 4.0. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

[0016] In some embodiments according to any of the embodiments described above, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml or 50 pg / ml.

[0017] In some embodiments according to any of the embodiments described above, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm. In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm.

[0018] In some embodiments, the lyoprotectant is a PVP and the buffer is PBS or a phosphate buffer. In some embodiments, the lyoprotectant is a PVP and the buffer is a Tris buffer. In some embodiments, the lyoprotectant is a PVP and the buffer is an acetate buffer. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

[0019] In some embodiments, the lyoprotectant is a PEG and the buffer is PBS or a phosphate buffer. In some embodiments, the lyoprotectant is a PEG and the buffer is a Tris buffer. In some embodiments, the lyoprotectant is a PEG and the buffer is an acetate buffer. In some embodiments, the PEG is PEG200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

[0020] In another aspect of the invention, there is provided a reconstituted formulation comprising a NA-LNP particle, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation according to any of the lyophilized formulations described above. In some embodiments according to the reconstituted formulation described above, the liquid is water or a buffer.

[0021] In another aspect of the invention, there is provided a method of making a lyophilized formulation comprising a NA-LNP particle, wherein the NA-LNP particle comprises anucleic acid encapsulated in the lipid nanoparticle, the method comprising: i) generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii) adding an excipient comprising a lyoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation; wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG); and iii) lyophilizing the aqueous NA-LNP formulation to produce the lyophilized formulation.

[0022] In some embodiments according to any of the methods described above, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0023] In some embodiments, any of the methods described above further comprises after step (ii) and before step (iii): a) freezing the aqueous NA-LNP formulation to obtain a frozen formulation, and b) removing water from the formulation by sublimation, wherein the freezing of step a) occurs at -20 degrees Celsius or -80 degrees Celsius.

[0024] In some embodiments according to any of the methods described above, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k.

[0025] In some embodiments according to any of the methods described above, the total concentration of the one or more lipids in the organic phase is between 0.1 mM to 20 mM. In some embodiments, the concentration of each of the one or more lipids in the organic phase is 0.1 mM, 0.125 mM, 0.2 mM, 0.5 mM, 1 mM, 6.25 mM, 10 mM, 12.5 mM, or 20 mM.

[0026] In some embodiments according to any of the methods described above, PVP is PVP- K12, PVP-K15, PVP-K30, PVP-K60, PVP-K90, or PVP-K120. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG- 8000, PEG-12000 or PEG-20000.

[0027] In some embodiments according to any of the methods described above, the nucleic acid comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA is a circular DNA or a linear DNA. In some embodiments, the DNA is a plasmid DNA. In some embodiments, the DNA is a genomic DNA. In some embodiments, the DNA is a synthetic DNA. In some embodiments, the synthetic DNA is a cDNA.

[0028] In some embodiments according to any of the methods described above, the nucleic acid comprises a ribonucleic acid (RNA). In some embodiments, the RNA is messenger RNA.

[0029] In some embodiments, the DNA or RNA is an antisense oligonucleotide.

[0030] In some embodiments according to any of the methods described above, the concentration of the lyoprotectant in step (ii) is between 5% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in step (ii) is 5%, 10%, 20% or 30% weight per volume (w / v). In some embodiments, the lyoprotectant is is 5% PVP- K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25. In some embodiments, the lyoprotectant is PEG, wherein the lyoprotectant is 10% PEG- 1500.

[0031] In some embodiments according to any of the methods described above, the buffer is an acetate buffer, and wherein the concentration of the acetate buffer in step (ii) is 10 mM, 20 mM or 50 mM. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0032] In some embodiments according to any of the methods described above, the buffer is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS or the buffer is a phosphate buffer. In some embodiments, the buffer is a Tris buffer, and wherein the concentration of the Tris buffer is 10 mM, 20 mM or 50 mM.

[0033] In some embodiments according to any of the methods described above, the pH of the buffer in step (ii) is between pH 4.0 and pH 8.0. In some embodiments, the pH of the buffer is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

[0034] In some embodiments according to any of the methods described above, the organic phase and the aqueous phase are mixed at a flow rate ratio of 1 :3 of organic: aqueous phase.

[0035] In some embodiments according to any of the methods described above, the method further comprises reconstituting the lyophilized formulation with a liquid to produce a reconstituted formulation comprising the NA-LNP particle. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is between 50 nm and 300 nm. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 10% larger, 20% larger or 30% larger than the diameter of the NA-LNP particle in the NA-LNP formulation in step ii). In some embodiments, the percent encapsulation efficiency (%EE) of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower, 20% lower, or 30% lower than the percent encapsulation efficiency (%EE) of the of the nucleic acid in the NA- LNP particle in the NA-LNP formulation in step ii). In some embodiments, the percent encapsulation efficiency (%EE) is calculated asSoluble nucleic acid concentration%EE = (1 - — - - - — - - - ; - ) x 100%Total nucleic acid concentrationIn some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower, 20% lower, or 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the NA-LNP formulation in step ii).

[0036] In another aspect of the invention, there is provided an in vitro or ex vivo method for introducing a nucleic acid encapsulated in a lipid nanoparticle into a cell, the method comprising contacting the cell with a reconstituted formulation, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation according to any of the lyophilized formulations described above with a liquid. In another aspect of the invention, there is provided a use of a reconstituted formulation comprising a nucleic acid encapsulated in a lipid nanoparticle in the manufacture of a medicament for use in a method of introducing the nucleic acid encapsulated in a lipid nanoparticle into a cell, wherein the method comprises contacting the cell with the reconstituted formulation, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation according to any of the lyophilized formulations described above with a liquid. In some embodiments, the liquid is water or a buffer. In some embodiments, the nucleic acid is expressed in the cells. In some embodiments, the cells do not produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.

[0037] In another aspect of the invention, there is provided a frozen formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a cryoprotectant and a buffer; wherein the cryoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG).

[0038] In some embodiments according to any of the frozen formulations described above, the PVP is PVP-K12, PVP -KI 5, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

[0039] In some embodiments according to any of the frozen formulations described above, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

[0040] In some embodiments according to any of the frozen formulations described above, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0041] In some embodiments according to any of the frozen formulations described above, the nucleic acid comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNAis a circular DNA or a linear DNA. In some embodiments, the DNA is a plasmid DNA. In some embodiments, the DNA is a genomic DNA. In some embodiments, the DNA is a synthetic DNA. In some embodiments, the synthetic DNA is a cDNA.

[0042] In some embodiments, the nucleic acid comprises a ribonucleic acid (RNA). In some embodiments, the RNA is a circular RNA or a linear RNA. In some embodiments, the RNA is messenger RNA. In some embodiments, the RNA is a micro RNA (miRNA). In some embodiments, the RNA is a small inhibitory RNA (siRNA). In some embodiments, the RNA is a guide RNA (gRNA).

[0043] In some embodiments, the DNA or RNA is an antisense oligonucleotide.

[0044] In some embodiments according to any of the frozen formulations described above, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises l,2-distearoyl-sn-glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k.

[0045] In some embodiments according to any of the frozen formulations described above, the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the cryoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v). In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the cryoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 20% or 30% weight per volume (w / v). In some embodiments, the cryoprotectant is is 5% PVP-K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP- K25. In some embodiments, the cryoprotectant is PEG, wherein the lyoprotectant is 10% PEG-1500.

[0046] In some embodiments according to any of the frozen formulations described above, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or the buffer is a phosphate buffer. In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM. In someembodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the frozen formulation is obtained from an aqueous NA- LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

[0047] In some embodiments according to any of the frozen formulations described above, the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is between pH 4.0 and pH 8.0. In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 4.0. In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

[0048] In some embodiments according to any of the frozen formulations described above, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the frozen formulation is obtained from an aqueous NA- LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml or 50 pg / ml.

[0049] In some embodiments according to any of the frozen formulations described above, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm. In some embodiments, the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm.

[0050] In some embodiments, the cryoprotectant is a PVP and the buffer is PBS or a phosphate buffer. In some embodiments, the cryoprotectant is a PVP and the buffer is a Tris buffer. In some embodiments, the cryoprotectant is a PVP and the buffer is an acetate buffer. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

[0051] In some embodiments, the cryoprotectant is a PEG and the buffer is PBS or a phosphate buffer. In some embodiments, the cryoprotectant is a PEG and the buffer is a Trisbuffer. In some embodiments, the cryoprotectant is a PEG and the buffer is an acetate buffer. In some embodiments, the PEG is PEG200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

[0052] In some embodiments, the frozen formulation is frozen at -20 degrees Celsius or -80 degrees Celsius.

[0053] In another aspect of the invention, there is provided a thawed formulation comprising a NA-LNP particle, wherein the thawed formulation is obtained by thawing the frozen formulation according to any of the frozen formulations described above.

[0054] In some embodiments according to any of the thawed formulations described above, the thawed formulation is obtained by at least one, at least two, or at least three cycles comprising (i) freezing the thawed NA-LNP formulation to obtain a frozen NA-LNP formulation, followed by (ii) thawing the frozen formulation of (i). In some embodiments, the freezing of (i) occurs at -20 degrees Celsius or -80 degrees Celsius to obtain a frozen formulation.

[0055] In some embodiments according to any of the thawed formulations described above, the frozen formulation is thawed at about 20 degrees Celsius to 25 degrees Celsius.

[0056] In another aspect of the invention, there is provided a method of making a frozen formulation comprising a NA-LNP particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, the method comprising: i) generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii) adding an excipient comprising a cryoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation; wherein the cryoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG); and iii) freezing the aqueous NA-LNP formulation to obtain a frozen formulation.

[0057] In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0058] In some embodiments, the freezing of step (iii) occurs at -20 degrees Celsius or -80 degrees Celsius.

[0059] In some embodiments, the method further comprises comprising: step (iv) thawing the frozen formulation to obtain a thawed formulation, then freezing the thawed formulation. In some embodiments, step (iv) is repeated at least one, at least two, or at least three times. In some embodiments, the thawing of step (iv) occurs at about 20 to 25 degrees Celsius.

[0060] In some embodiments, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k. In some embodiments, the total concentration of the one or more lipids in the organic phase is between 0.1 mM to 20 mM. In some embodiments, the concentration of each of the one or more lipids in the organic phase is 0.1 mM, 0.125 mM, 0.2 mM, 0.5 mM, 1 mM, 6.25 mM, 10 mM, 12.5 mM, or 20 mM.

[0061] In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K30, PVP-K60, PVP- K90, or PVP-K120.

[0062] In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG- 1500, PEG- 3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

[0063] In some embodiments, the nucleic acid comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA is a circular DNA or a linear DNA. In some embodiments, the DNA is a plasmid DNA. In some embodiments, the DNA is a genomic DNA. In some embodiments, the DNA is a synthetic DNA. In some embodiments, the synthetic DNA is a cDNA.

[0064] In some embodiments, the nucleic acid comprises a ribonucleic acid (RNA). In some embodiments, the RNA is messenger RNA.

[0065] In some embodiments, the DNA or RNA is an antisense oligonucleotide.

[0066] In some embodiments, the concentration of the cryoprotectant in step (ii) is between 5% and 40% weight per volume (w / v). In some embodiments, the concentration of the cryoprotectant in step (ii) is 5%, 10%, 20% or 30% weight per volume (w / v). In some embodiments, the cryoprotectant is is 5% PVP-K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25. In some embodiments, the cryoprotectant is 10% PEG- 1500.

[0067] In some embodiments, the buffer is an acetate buffer, and wherein the concentration of the acetate buffer in step (ii) is 10 mM, 20 mM or 50 mM. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the buffer is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or the buffer is a phosphate buffer. In some embodiments, the buffer is a Tris buffer, and wherein the concentration of the Tris buffer is 10 mM, 20 mM or 50 mM.

[0068] In some embodiments, the pH of the buffer in step (ii) is between pH 4.0 and pH 8.0. In some embodiments, the pH of the buffer is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

[0069] In some embodiments, the organic phase and the aqueous phase are mixed at a flow rate ratio of 3 : 1.

[0070] In some embodiments, the method further comprises thawing the frozen formulation to produce a thawed formulation comprising the NA-LNP particle. In some embodiments, the diameter of the NA-LNP particle in the thawed formulation is between 50 nm and 300 nm. In some embodiments, the diameter of the NA-LNP particle in the thawed formulation is about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the thawed formulation is no more than 10% larger, 20% larger or 30% larger than the diameter of the NA-LNP particle in the NA-LNP formulation in step ii). In some embodiments, the percent encapsulation efficiency (%EE) of the nucleic acid in the NA-LNP particle in the thawed formulation is no more than 10% lower, 20% lower, or 30% lower than the percent encapsulation efficiency (%EE) of the of the nucleic acid in the NA-LNP particle in the NA- LNP formulation in step ii). In some embodiments, the percent encapsulation efficiency (%EE) is calculated asSoluble nucleic acid concentration%EE = (1 - — - - - — - - -;- ) x 100%Total nucleic acid concentration

[0071] In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the thawed formulation is no more than 10% lower, 20% lower, or 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the NA-LNP formulation in step ii).

[0072] In another aspect of the invention, there is provided an in vitro or ex vivo method for introducing a nucleic acid encapsulated in a lipid nanoparticle into a cell, the method comprising contacting the cell with a thawed formulation, wherein the aqueous formulation is obtained by thawing the frozen formulation according to any of the frozen formulations described above. In another aspect of the invention, there is provided a use of a thawed formulation comprising a nucleic acid encapsulated in a lipid nanoparticle in the manufacture of a medicament for use in a method of introducing the nucleic acid encapsulated in a lipid nanoparticle into a cell, wherein the method comprises contacting the cell with the thawed formulation, wherein the thawed formulation is obtained by thawing the frozen formulation according to any of the frozen formulations described above. In some embodiments, the frozen formulation is thawed at 20 degrees Celsius to 25 degrees Celsius. In some embodiments, the nucleic acid is expressed in the cells. In some embodiments, the cells donot produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.BRIEF DESCRIPTION OF THE DRAWINGS

[0073] The drawings illustrate certain embodiments of the features of this disclosure. These embodiments are not intended to limit the scope of the claims in any manner.

[0074] FIG. 1 shows a scheme of the experimental strategy for high-throughput screening of lyoprotectant and buffer conditions, and establishment of the structure-activity relationship (SAR) based on the physicochemical and formulation efficacy characterizations of the screened formulations.

[0075] FIG. 2 shows the cake appearance of lyophilized mRNA-LNP formulations with 10% of different excipients and in different buffers.

[0076] FIG. 3 shows particle size changes of lyophilized mRNA-LNPs with different lyoprotectant and buffer conditions.

[0077] FIG. 4 shows changes in the percent encapsulation efficiency (%EE) of mRNA in lyophilized mRNA-LNPs with different lyoprotectant and buffer conditions.

[0078] FIG. 5 shows changes in the percent of intact mRNA in lyophilized mRNA-LNPs with different lyoprotectant and buffer conditions.

[0079] FIG. 6 shows representative mRNA fragment analysis plots for the results shown in FIG. 5. Fresh mRNA-LNPs and reconstituted, lyophilized mRNA-LNPs were in Tris buffer with 10% of different excipients. The percent intact mRNA is presented as mean ± SD from two technical replicates.

[0080] FIGS. 7A-7B show in vitro activity of lyophilized mRNA-LNPs tested in HEK-293T cells, shown as changes of the frequency of EGFP+population (FIG. 7A) and fold changes of geometric mean fluorescence intensity (gMFI) over the fresh formulation control (FIG. 7B). Lyophilization with PVP-K12 or PEG-1500 in PBS resulted in significant decrease of in vitro activity; whereas some sucrose and maltose conditions showed higher gMFI values than their corresponding formulations before lyophilization. Fresh mRNA-LNPs showed translation efficiency of 76.7 ± 1.2%, and the mean value was subtracted from individual sample result values to calculate the percent changes shown in FIG. 7A.

[0081] FIG. 8 shows a comparison of in vitro activity of lyophilized mRNA-LNPs (lyophilized mRNA-LNPs with 10% of individual excipient and different buffers) tested in human monocytes and HEK-293T cells. Fold changes of gMFI over the results of fresh mRNA-LNPs in the respective plain buffers showed a similar rank ordering of lyoprotectant performance between the two cell lines. Monocyte results are compiled from three differentPBMC donors. HEK-293T data with two technical replicates is reproduced from the study shown in FIGS. 7A-7B.

[0082] FIG. 9 shows a heatmap of cytokine secretion from human PBMCs treated with fresh or lyophilized mRNA-LNPs under different lyoprotectant and buffer conditions. Results are compiled from three different PBMC donors.

[0083] FIGS. 10A-10C show correlations between changes of physicochemical properties, (FIG. 10A) particle size, (FIG. 10B) %EE of mRNA, (FIG. 10C) mRNA integrity, and in vitro activity. Analyses were performed using the same data set shown in FIGS. 3-5 and FIG. 7. The PVP-K12 in PBS conditions, which resulted in significant aggregates, were excluded from the analysis in FIG. 10A.

[0084] FIGS. 11A-11F show correlations among physicochemical property changes of mRNA-LNPs after lyophilization, including (FIG. 11 A) particle size vs. %EE of mRNA, (FIG. 11B) particle size vs. mRNA integrity, (FIG. 11C) %EE of mRNA vs. mRNA integrity, (FIG. 11D) particle size vs. LNP ordered phase area, (FIG. HE) %EE of mRNA vs. LNP ordered phase area, and (FIG. HF) mRNA integrity vs. LNP ordered phase area. LNP physical instability and mRNA chemical instability were correlated. Further, changes in particle size, %EE of mRNA and mRNA integrity were all correlated with the percent change of total ordered LNP core structures. Data were reprocessed from those shown in FIGS. 3-5 and 12. The PVP-K12 in PBS conditions, which resulted in significant aggregates, were excluded from analyses involving particle size changes. Lysine conditions were excluded from analyses involving total ordered phases. Data are shown by mean ± SD, or only mean values for analyses related to LNP structures.

[0085] FIG. 12 shows the relationship between mRNA-LNP structure and in vitro activity changes after lyophilization. LNP structures are quantified by the percent changes in the total ordered (Hu and La) core phase areas, compared with data from the corresponding fresh formulation. Activity change data is reprocessed from FIG. 7A. The shaded area of the % change of LNP ordered phase area plot indicates the minimal activity change region of ±10% compared with the fresh formulation. Deconvolved SAXS data and the cryo-TEM images are also shown for representative formulation conditions in each quadrant: (I) total structural order increases, activity remains similar; (II) total structural order decreases, activity remains similar or slightly increases; and (III) total structural order decreases, activity also decreases. The conditions near the origin point represent samples whose total structural order and activity are both similar to those of the fresh formulation. Scale bar in cryo-TEM images, 100 nm.

[0086] FIG. 13 shows structural analyses of additional representative samples, with their data points also highlighted in FIG. 12. For SAXS profiles after lyophilization in PBS, the cumulative fitting mostly overlaps with the curve representing the disordered feature, suggesting the ordered features are almost lost. Scale bar in cryo-TEM images, 100 nm.

[0087] FIG. 14 shows total ordered core phase areas quantified for lyophilized mRNA-LNPs in different buffers. Results are calculated from the deconvolved SAXS data and presented as box whisker plots to indicate the min, median, and max levels. **P < 0.01, ****P < 0.0001, ns, not significant, analyzed by one-way ANOVA followed by Tukey's test for multiple comparisons.

[0088] FIG. 15 shows particle size changes of mRNA-LNPs after three freeze / thaw cycles with different lyoprotectant and buffer conditions.

[0089] FIG. 16 shows changes in the percent encapsulation efficiency (%EE) of mRNA in mRNA-LNPs after three freeze / thaw cycles with different lyoprotectant and buffer conditions.

[0090] FIG. 17 shows changes in the percent of intact mRNA in mRNA-LNPs after three freeze / thaw cycles with different lyoprotectant and buffer conditions.

[0091] FIG. 18 shows in vitro activity of mRNA-LNPs tested in HEK-293T cells after three freeze / thaw cycles, shown as changes of the frequency of EGFP+population.

[0092] FIGS. 19A-19C show correlations between changes of physicochemical properties of mRNA-LNPs after three cycles of freeze-thaw, each of (FIG. 19A) particle size, (FIG. 19B) %EE of mRNA, or (FIG. 19C) mRNA integrity, and in vitro activity changes after three freeze / thaw cycles. Analyses were performed using the same data set shown in FIGS. 15-18.

[0093] FIGS. 20A-20F show correlations among physicochemical property changes of mRNA-LNPs after three cycles of freeze-thaw, including (FIG. 20A) particle size vs. %EE of mRNA, (FIG. 20B) particle size vs. mRNA integrity, (FIG. 20C) %EE of mRNA vs. mRNA integrity, (FIG. 20D) particle size vs. LNP ordered phase area, (FIG. 20E) %EE of mRNA vs. LNP ordered phase area, and (FIG. 20F) mRNA integrity vs. LNP ordered phase area. Data were reprocessed from those shown in FIGS. 15-17 and 21. Data are shown by mean ± SD, or only mean values for analyses related to LNP structures.

[0094] FIG. 21 shows the relationship between mRNA-LNP structure and in vitro activity changes after three cycles of freeze-thaw. LNP structures are quantified by the percent changes in the total ordered (Hu and La) core phase areas, compared with data from the corresponding fresh formulation. Activity change data is reproduced from FIG. 18. Theshaded area indicates the minimal activity change region of ±10% compared with the fresh formulation.

[0095] FIG. 22 shows particle size changes of lyophilized anti-sense oligonucleotide (ASO)- LNPs after reconstitution (black bars) and ASO-LNPs after one freeze / thaw cycle (white bars), each with different lyoprotectant or cryoprotectant conditions in phosphate buffer (10 mM, pH 7.4).

[0096] FIG. 23 shows changes in the percent encapsulation efficiency (%EE) of ASO in lyophilized ASO-LNPs (black bars) and freeze-thaw ASO-LNPs (white bars) with different lyoprotectant and cryoprotectant conditions in phosphate buffer (10 mM, pH 7.4).

[0097] FIG. 24 shows correlations between changes in particle size vs. %EE of ASO-LNPs after one cycle of freeze-thaw (FIG. 24A) or after lyophilization (FIG. 24B). Data were reprocessed from those shown in FIGS. 22-23.

[0098] FIG. 25 shows the particle size change of mRNA-LNPs after lyophilization and reconstitution. EGFP mRNA-LNPs were prepared in 10 mM Tris buffer (pH 7.5) supplemented with different PVP excipients. Size changes were calculated by subtracting individual fresh sample mean diameter from the corresponding sample mean diameter after lyophilization.

[0099] FIG. 26 shows changes in the percent encapsulation efficiency (%EE) of mRNA in mRNA-LNPs after lyophilization and reconstitution. EGFP mRNA-LNPs were prepared in 10 mM Tris buffer (pH 7.5) supplemented with different PVP excipients. %EE changes were calculated by subtracting the %EE of the fresh sample (93.8% before spiking in excipients) from individual sample %EE after lyophilization.DETAILED DESCRIPTION

[0100] A diverse range of NA-LNP vaccines are now undergoing clinical trials. Beyond infectious diseases, the scope of therapeutic range is expanding to encompass genetic disorders and cancers. The primary challenge faced by the NA-LNP platform is the necessity for a cold-chain system for logistics and long-term storage. The demand for cold chain storage presents significant challenges to NA-LNP therapeutic distribution in countries with insufficient infrastructure.

[0101] Ultra-cold storage is essential for preserving the integrity and efficacy of NA-LNP due to multiple factors. Primarily, the NA cargo is inherently fragile and prone to rapiddegradation especially in the solution state and elevated temperatures. See, L. Schoenmaker, et al., Int J Pharm, 601 (2021) 120586; U. Chheda, et al., J Pharm Sci, 113 (2024) 377-385). Additionally, the LNP carrier, assembled by multiple lipid components, exhibits complex intermolecular interactions and is sensitive to environmental changes. The ultra-cold conditions prevent aggregation of LNPs upon storage and maintain the levels of encapsulated NAs. Exploring an alternative, the lyophilization (freeze-drying) approach emerges as a promising strategy to extend the shelf life while eliminating the need for ultra-cold logistics by removing the water from drug formulations. Throughout the lyophilization process, NA- LNPs face various stressors, including but not limited to crystallization, alterations in ionic strength, and interfacial stress between ice and liquid. The stresses induced during lyophilization may introduce variations in formulation quality attributes, such as particle size, cargo encapsulation, in vitro activity, and LNP structures.

[0102] The present invention is at least partly based upon the surprising stability of a lyophilized NA-LNP formulation using polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG) as the lyoprotectant. In particular, PVP-K12 and PEG-1500 preferably maintain physicochemical stability and functionality of lyophilized NA-LNPs, as assessed by measures such as change in particular size, change in NA encapsulation, change in NA integrity, change in in vitro activity of the NA-LNPs after lyophilization, and change in NA translation efficiency.

[0103] Provided herein, in certain aspects, are lyophilized formulations comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant and a buffer, and wherein the lyoprotectant is polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Also provided herein, in certain aspects, are reconstituted formulations comprising a NA-LNP particle, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulations with a liquid. Also provided herein, in other aspects, are methods of making a lyophilized formulation comprising a NA-LNP particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, the method comprising: i) generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii) adding an excipient comprising a lyoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation, wherein the lyoprotectant is polyvinylpyrrolidone(PVP) or polyethylene glycol (PEG); and iii) lyophilizing the aqueous NA-LNP formulation to produce the lyophilized formulation.I. Definitions

[0104] Before describing the invention in detail, it is to be understood that this invention is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0105] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless indicated otherwise. For example, “a disorder” includes one or more disorders.

[0106] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”.

[0107] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat disease of type X means the method is used to treat disease of types other than X.

[0108] The term “about X-Y” used herein has the same meaning as “about X to about Y ”

[0109] The phrase “comprising” as used herein is open-ended, indicating that such embodiments may include additional elements. In contrast, the phrase “consisting of’ is closed, indicating that such embodiments do not include additional elements (except for trace impurities). The phrase “consisting essentially of’ is partially closed, indicating that such embodiments may further comprise elements that do not materially change the basic characteristics of such embodiments. It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0110] As used herein, a “baseline” level in a human refers to the level before an administration of a drug (e.g., NA-LNP therapeutic) described herein to the human. [OHl] As used herein, the term “prevention” includes providing prophylaxis with respect to occurrence or recurrence of a disorder in an individual (e.g., human subject).

[0112] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations are sterile. “Pharmaceutically acceptable”excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0113] A “pharmaceutically acceptable carrier” refers to one or more ingredients in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, lyoprotectant, stabilizer, cryoprotectant, tonicity agent, preservative, and combinations thereof.

[0114] The terms “pre-lyophilized formulation” and “lyophilizable formulation” are used herein interchangeably to refer to formulations that are subject to lyophilization to prepare a lyophilized formulation.

[0115] A “stable” formulation is one in which the protein therein essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. In some embodiments, the formulation essentially retains its physical and chemical stability, as well as its biological activity upon storage. The storage period is generally selected based on the intended shelf-life of the formulation. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247- 301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can be measured at a selected amount of light exposure and / or temperature for a selected time period. Stability can be evaluated qualitatively and / or quantitatively in a variety of different ways, including, but not limited to, evaluation of change in particular size, change in NA encapsulation, change in NA integrity, and change in in vitro activity of the NA-LNPs after lyophilization, and change in NA translation efficiency; evaluation of aggregate formation (for example using size exclusion chromatography, by measuring turbidity, and / or by visual inspection); evaluation of ROS formation (for example by using a light stress assay or a 2,2’ -Azobis(2- Amidinopropane) Dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues of the protein (for example a Trp residue and / or a Met residue of a monoclonal antibody); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometric analysis; SDS-PAGE analysis to compare reduced and intact antibody; peptide map (for example tryptic or LYS-C) analysis; evaluating biological activity or target binding function of the protein (e.g., antigen binding function of an antibody); etc. Instability may involve any one or more of: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and / or Trp oxidation),isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C- terminal processing, glycosylation differences, degradation of excipients, formation of particulates e.g., free fatty acid particles), etc.

[0116] As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with the disease (e.g., asthma) are mitigated or eliminated, including, but are not limited to, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and / or prolonging survival of individuals.

[0117] An “effective amount” is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including, but not limited to, biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and / or prolonging survival. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.

[0118] As used herein, “in conjunction with” or “in combination with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” or “in combination with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.

[0119] A “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question.

[0120] A “subject”, “patient” or an “individual” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human.

[0121] As used herein, a “nucleic acid” is preferably a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably RNA, most preferably an mRNA. Nucleic acids include, but are not limited to, genomic DNA, cDNA, mRNA, micro RNA (miRNA), small inhibitory RNA (siRNA), guide RNA (gRNA), recombinantly produced and chemically synthesized molecules. A nucleic acid may according to the invention be in the form of a molecule which is single stranded or double stranded and linear or closed covalently to form a circle. A nucleic can be employed for introduction into, i.e. transfection of, cells, for example, in the form of RNA which can be prepared by in vitro transcription from a DNA template. The RNA can moreover be modified before application by stabilizing sequences, capping, and polyadenylation.

[0122] As used herein, the term “nucleic acid” refers to polymeric forms of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.

[0123] As used herein, the term “DNA” relates to a molecule which comprises deoxyribonucleic acid and preferably being entirely or substantially composed of deoxyribonucleic acid residues.

[0124] As used herein, the term “RNA” relates to a molecule which comprises ribonucleotide residues and preferably being entirely or substantially composed of ribonucleotide residues. “Ribonucleotide” relates to a nucleotide with a hydroxyl group at the 2'-position of a P-D- ribofuranosyl group. The term includes, but is not limited to, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of an RNA or internally, for example at one or more nucleotides of the RNA. Nucleotides in RNA molecules can also comprise non-standard nucleotides, such as non- naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs or analogs of naturally occurring RNA. The term “RNA” includes “mRNA” which means “messenger RNA” and relates to a “transcript” which may be produced using DNA as template and encodes a peptide or protein. mRNA typically comprises a 5' non translated region (5'-UTR), a protein or peptide coding region and a 3' non translated region (3'-UTR). mRNA has a limited halftime in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available.

[0125] As used herein, the term “translation” relates to the process in the ribosomes of a cell by which a strand of messenger RNA directs the assembly of a sequence of amino acids to make a protein or peptide.

[0126] The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies (including but not limited to full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0127] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the compositions and methods described herein are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitlydisclosed. In addition, all subcombinations of features and properties of the compositions and methods in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.II. Lyophilized formulations

[0128] The present invention relates to lyophilized formulations comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle. The NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle. The formulation further comprises an excipient comprising a lyoprotectant and a buffer. The lyoprotectant is polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). In some embodiments the buffer is phosphate-buffered saline (PBS). In some embodiments the buffer is phosphate buffer. In some embodiments the buffer is Tris. In some embodiments the buffer is an acetate buffer.

[0129] The terms “lyophilization,” “lyophilized,” and “freeze-dried” can refer, for example, to a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapor without passing through a liquid phase. The terms “lyophilization,” “lyophilized,” and “freeze-dried” can also refer, for example, to a process by which the material to be is dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment.

[0130] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises PVP. PVP is also known as polyvidone or povidone. PVP is a polymer molecule which comprises the monomer N-vinylpyrrolidone. In some embodiments, the PVP is entirely or substantially composed of N-vinylpyrrolidone. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP- K60, or PVP-K90. In some embodiments the PVP is low molecular weight PVP. In some embodiments, the low molecular weight PVP is PVP-K12, PVP-K15, or PVP-K17. In some embodiments, the PVP is PVP-K12. In some embodiments the PVP is medium molecular weight PVP. In some embodiments, the medium molecular weight PVP is PVP-K25 or PVP- K30. In some embodiments the PVP is high molecular weight PVP. In some embodiments, the high molecular weight PVP is PVP-K60 or PVP-K90.

[0131] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises PEG. In some embodiments, the PEG is PEG- 200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is a low molecular weight PEG. In some embodiments, the low molecular weight PEG is PEG-200, PEG-300, PEG-400, PEG- 600, or PEG-1500. In some embodiments, the PEG is PEG-1500. In some embodiments, PEG-1500 comprises PEG-300 or PEG1540. In some embodiments, the PEG is a medium molecular weight PEG. In some embodiments, the medium molecular weight PEG is PEG- 1000, PEG-1540, PEG-2000. In some embodiments, the PEG is a high molecular weight PEG. In some embodiments, the high molecular weight PEG is PEG-3100, PEG-3400, PEG- 8300, PEG-8600, PEG-11000, PEG-13000, or PEG-20000.

[0132] The lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises a buffer. As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In some embodiments, the buffer of this invention has a pH from about 4.0 to about 8.0, for example from about 5.0 to about 7.0, e.g. from about 5.8 to about 6.2. Examples of buffers include, but are not limited to, acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In one embodiment herein, the buffer is a histidine buffer. In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0133] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises a phosphate-buffered saline (PBS) buffer. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline.

[0134] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises a Tris buffer. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris is Tris-Boric Acid-EDTA(TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE). In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris has a pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3.

[0135] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises an acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In some embodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0136] The lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PEG is PEG- 200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG- 12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0137] The lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PVP is PVP- K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0138] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises an oligosaccharide. The lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is PBS. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is an acetate buffer. In some embodiments, the oligosaccharide is sucrose, maltose, trehalose, lactose, glucose, or any combination thereof. In some embodiments, the oligosaccharide is sucrose. In some embodiments, the oligosaccharide is maltose. In some embodiments, the oligosaccharide is trehalose. In some embodiments, the oligosaccharide is lactose. In some embodiments, the oligosaccharide is glucose.

[0139] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises sucrose. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 0%-40%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-l 0%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 10%- 15%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 15%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 20%-25%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 25%-30%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 30%-35%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 35%-40%. In some embodiments, the concentration of sucrose is 5%. In some embodiments, the concentration of sucrose is 10%. In some embodiments, the concentration of sucrose is 15%. In some embodiments, the concentration of sucrose is 20%. In some embodiments, the concentration of sucrose is 25%. In some embodiments, the concentration of sucrose is 30%. In some embodiments, the concentration of sucrose is 35%. In some embodiments, the concentration of sucrose is 40%.

[0140] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises maltose. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 0%-40%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-l 0%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises maltose at a concentration of 10%-l 5%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 15%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 20%-25%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 25%-30%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 30%-35%. In some embodiments, the lyophilized formulation comprising anucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 35%-40%. In some embodiments, the concentration of maltose is 5%. In some embodiments, the concentration of maltose is 10%. In some embodiments, the concentration of maltose is 15%. In some embodiments, the concentration of maltose is 20%. In some embodiments, the concentration of maltose is 25%. In some embodiments, the concentration of maltose is 30%. In some embodiments, the concentration of maltose is 35%. In some embodiments, the concentration of maltose is 40%.

[0141] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 0%-40%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 5%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 5%-l 0%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 10%- 15%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 15%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 20%-25%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 25%-30%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 30%-35%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 35%-40%. In some embodiments, the concentration of trehalose or lactose or glucose is 5%. In some embodiments, the concentration of trehalose or lactose or glucose is 10%. In some embodiments, the concentration of trehalose or lactose or glucose is 15%. In some embodiments, the concentration of trehalose or lactose or glucose is 20%. In some embodiments, the concentration of trehalose or lactose or glucose is 25%. In someembodiments, the concentration of trehalose or lactose or glucose is 30%. In some embodiments, the concentration of trehalose or lactose or glucose is 35%. In some embodiments, the concentration of trehalose or lactose or glucose is 40%.

[0142] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises an amino acid. The lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an amino acid. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is PBS. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is an acetate buffer. In some embodiments, the amino acid is lysine, arginine, leucine, glutamic acid, or any combination thereof. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is arginine. In some embodiments, the amino acid is leucine. In some embodiments, the amino acid is glutamic acid.

[0143] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises lysine. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 0%-40%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-l 0%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises lysine at a concentration of 10%- 15%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 15%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 20%-25%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 25%-30%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 30%-35%. In some embodiments, the lyophilized formulation comprising anucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 35%-40%. In some embodiments, the concentration of lysine is 5%. In some embodiments, the concentration of lysine is 10%. In some embodiments, the concentration of lysine is 15%. In some embodiments, the concentration of lysine is 20%. In some embodiments, the concentration of lysine is 25%. In some embodiments, the concentration of lysine is 30%. In some embodiments, the concentration of lysine is 35%. In some embodiments, the concentration of lysine is 40%.

[0144] In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 0%-40%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 5%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 5%-10%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 10%-l 5%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 15%-20%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 20%-25%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 25%-30%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 30%-35%. In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 35%-40%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 5%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 10%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 15%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 20%. In some embodiments, the concentration of arginine or leucine orglutamic acid is 25%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 30%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 35%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 40%.

[0145] In some embodiments, the lyophilized formulation comprises a deoxyribonucleic acid (DNA). In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising a DNA. In some embodiments, the DNA is a singlestranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. In some embodiments, the DNA is a plasmid, oligonucleotide, antisense oligonucleotide (ASO), DNA aptamer, or DNAzyme. In some embodiments, the DNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15- 20 nucleotides in length. In some embodiments, the ASO binds a complementary RNA target. In some embodiments, the DNA is circular. In some embodiments, the DNA is linear. In some embodiments, the DNA is copy DNA or complementary DNA (cDNA). In some embodiments, the DNA is a synthetic DNA. In some embodiments, the DNA is a PCR amplicon. In some embodiments, the DNA is synthesized by endonuclease-mediated assembly, BioBricks, Golden Gate cloning, or Gibson assembly. In some embodiments, the DNA is a naturally occurring DNA. In some embodiments, the DNA is extracted from a cell or tissue. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / mland 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml.

[0146] In some embodiments, the lyophilized formulation comprises a ribonucleic acid (RNA). In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation comprising an RNA. In some embodiments, the RNA is a circular RNA or a linear RNA. In some embodiments, the RNA is linear RNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is a messenger RNA (mRNA). In some embodiments, the RNA is an engineered RNA. In some embodiments, the RNA is a naturally occurring RNA. In some embodiments, the RNA is a non-capped mRNA, a nonpolyadenylated mRNA, and a non-spliced mRNA. In some embodiments, the RNA is noncoding RNAs (ncRNA) that is not translated into a protein, e.g., small non-coding RNAs suchas microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), CRISPR RNA (crRNA), tracrRNA, guide RNA gRNA), and extracellular RNA (exRNA). In some embodiments, the RNA is a micro RNA (miRNA). In some embodiments, the RNA is a small inhibitory RNA (siRNA). In some embodiments, the RNA is a CRISPR-associated RNA, e.g., gRNA, crRNA, tracrRNA. In some embodiments, the RNA is a guide RNA (gRNA). In some embodiments, the RNA is small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA- derived RNA (srRNA). The RNA can be double-stranded RNA or single-stranded RNA. In some embodiments, the RNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15- 20 nucleotides in length. In some embodiments, the ASO binds a complementary RNA target. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration ofthe nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml.

[0147] As used herein, “LNPs” refer to “lipid nanoparticles”, which are nanoparticles comprising lipids. In some embodiments, the LNP is spherical. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm and about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm to about 75 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 75 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 100 nm to about 125 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 90 nm to about 110 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 125 nm to about 150 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 150 nm to about 200 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 200 nm to about 250 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 250 nm to about 300 nm. In some embodiments, thediameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 1000 nm. In some embodiments, the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 50 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm to about 200 nm. In some embodiments, the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 200 nm to about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 300 nm to about 400 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 400 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 500 nm to about 600 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 600 nm to about 700 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 700 nm to about 800 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 800 nm to about 900 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 900 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm, 25 nm, 50 nm, 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0148] As used herein, “LNPs” refer to “lipid nanoparticles”, which are nanoparticles comprising lipids. In some embodiments, the LNP is spherical. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 50 nm and about 300 nm. In some embodiments, the lyophilizedformulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 50 nm to about 75 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 75 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 100 nm to about 125 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 90 nm to about 110 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 125 nm to about 150 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 150 nm to about 200 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 200 nm to about 250 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 250 nm to about 300 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 50 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 100 nm to about 200 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 200 nm to about 300 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 300 nm to about 400 nm. In some embodiments, the lyophilized formulation comprises NA-LNPparticles, and the diameter of the NA-LNP particle is about 400 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 500 nm to about 600 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 600 nm to about 700 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 700 nm to about 800 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 800 nm to about 900 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 900 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm, 25 nm, 50 nm, 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0149] LNP structure is defined by its core, inner shell, and outer shell. The nucleic acid payload is encapsulated in a liquid crystalline phase in the core. The inner and outer shell are comprised of lipids. In some embodiments, the nanoparticles comprise at least one lipid. In some embodiments, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises 1,2-di stearoyl -sn- glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k. In some embodiments, the LNP comprises cholesterol. In some embodiments, the LNP comprises an ionizable lipid. In some embodiments, the LNP comprises a PEGylated lipid. In some embodiments, the LNP comprises cholesterol and an ionizable lipid. An ionizable lipid is a lipid that is positive at acidic pH and neutral at basic pH. Examples of ionizable lipids include ALC-0315, C12-200, cKK-E12, and DLin-MC3-DMA. In some embodiments, the LNP comprises cholesterol, an ionizable lipid, and DSPC. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the nucleic acid. In one embodiment, the at least one cationic lipid comprises l,2-di-0-octadecenyl-3-trimethylammonium propane (DOTMA) or analogs or derivatives thereof and / or 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof. In one embodiment, the nanoparticles comprise at least one helper lipid. Helper lipids are a class of lipid molecules that increase particle stability and fluidity of lipid nanoparticles (LNP). The helper lipid may be a neutral or an anionic lipid. Examples of helper lipids include DSPC, cholesterol, and PEGylated lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In some embodiments, the cationic lipid and / or the helper lipid is a bilayer forming lipid. In one embodiment, the at least one helper lipid comprises 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and / or 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC) or analogs or derivatives thereof. In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1 :2, 4: 1 to 2:3, 7:3 to 1 : 1, or 2: 1 to 1 : 1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm.

[0150] The lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising a lyoprotectant. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation and the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 10%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 15%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 20%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and 25%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 30%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 15% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation isbetween 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 30% and 40% weight per volume (w / v). In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the concentration of the lyoprotectant in the aqueous NA- LNP formulation is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, or 90% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 5% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 10% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 45% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 50% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 55% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 60% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 65% weight per volume (w / v). In some embodiments, the concentrationof the lyoprotectant in the aqueous NA-LNP formulation is about 70% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 75% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 80% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 90% weight per volume (w / v). In some embodiments, the lyoprotectant is PVP. In other embodiments, the lyoprotectant is PEG. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG- 20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0151] In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation comprising PBS. In some embodiments, the PBS in the aqueous NA-LNP formulation is lx PBS, 5x PBS or lOx PBS. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH ofabout 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is PBS.

[0152] In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation comprising a phosphate buffer. In some embodiments, the phosphate buffer has a pH of about 7.0 to about 9.0. In some embodiments, the phosphate buffer has a pH of about 7.0 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.8. In some embodiments, the phosphate buffer has a pH of about 8.0 to about 8.5. In some embodiments, the phosphate buffer has a pH of about 8.5 to about 9.0. In some embodiments, the phosphate buffer has a pH of about 7.4. In some embodiments, the phosphate buffer has a pH of about 7.5. In some embodiments, the phosphate buffer has a pH of about 7.6. In some embodiments, the phosphate buffer has a pH of about 8.0. In some embodiments, the phosphate buffer has a pH of about 8.3. In some embodiments, the concentration of the phosphate buffer in the aqueous formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 5 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA- LNP formulation is 150 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 200 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 250 mM. In some embodiments, theconcentration of phosphate buffer in the aqueous NA-LNP formulation is 300 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 400 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA- LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP- K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP- K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG- 3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer.

[0153] In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation comprising Tris. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 150 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 200 mM and 300 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 350 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 300 mM and 400 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 350 mM and 450 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 400 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 50 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 20 mM. In some embodiments, the concentrationof Tris in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 45 mM and 55 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 70 mM and 80 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 80 mM and 90 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA- LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 75 mM and 125 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, or IM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris is Tris-Boric Acid-EDTA (TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE) In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris has a pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP- K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG- 3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is Tris.

[0154] In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation comprising an acetate buffer. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 150 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 150 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 250 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 250 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 350 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 350 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 450 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 450 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of acetate buffer in theaqueous NA-LNP formulation is between 10 mM and 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 70 mM and 80 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 80 mM and 90 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 110 mM and 120 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA- LNP formulation is between 120 mM and 130 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 130 mM and 140 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 140 mM and 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 5 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 200 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 250 mM. In some embodiments, the concentration of acetatebuffer in the aqueous NA-LNP formulation is 300 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 400 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP- K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In some embodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0155] In some embodiments, the lyophilized formulation is obtained from an aqueous NA- LNP formulation, and the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 2.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 2.0 and about pH 3.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 3.0 and about pH 4.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.0 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 7.0. Insome embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 9.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 9.0 and about pH 10.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 10.0 and about pH 11.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 11.0 and about pH 12.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 12.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 4.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.5 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.0 and about pH 5.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.5 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 6.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.3 and about pH 6.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.4 and about pH 6.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.5 and about pH 7.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.7 and about pH 7.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.2 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.4 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.8 and about pH 8.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.5 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 8.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.2, pH 8.4, or pH 8.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0. In some embodiments, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is aphosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0156] The lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising a nucleic acid. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNPformulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA is mRNA.III. Reconstituted formulations

[0157] In one embodiment, the invention provides a reconstituted formulation comprising a NA-LNP particle. The reconstituted formulation is obtained by reconstituting a lyophilized formulation with a liquid. A “reconstituted” formulation can be a formulation which has been prepared by dissolving a lyophilized protein or antibody formulation in a liquid such that the protein is dispersed in the reconstituted formulation. In some embodiments, the liquid is water. In other embodiments, the liquid is a buffer. In some embodiments, the buffer is a histidine buffer, PBS, a phosphate buffer, a Tris buffer, or an acetate buffer. In some embodiments, the reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient to be treated with the NA-LNP.

[0158] In some embodiment, the reconstituted formulation is obtained by reconstituting a lyophilized formulation with water. In some embodiment, the reconstituted formulation is obtained by reconstituting a lyophilized formulation with a buffer. As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In some embodiments, the buffer has a pH from about 4.0 to about 8.0, for example from about 5.0 to about 7.0, e.g. from about 5.8 to about 6.2. Examples of buffers include, but are not limited to, acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In one embodiment herein, the buffer is ahistidine buffer. In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0159] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising PVP. PVP is also known as polyvidone or povidone. PVP is a polymer molecule which comprises the monomer N-vinylpyrrolidone. In some embodiments, the PVP is entirely or substantially composed of N-vinylpyrrolidone. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments the PVP is low molecular weight PVP. In some embodiments, the low molecular weight PVP is PVP-K12, PVP-K15, or PVP-K17. In some embodiments, the PVP is PVP-K12. In some embodiments the PVP is medium molecular weight PVP. In some embodiments, the medium molecular weight PVP is PVP-K25 or PVP- K30. In some embodiments the PVP is high molecular weight PVP. In some embodiments, the high molecular weight PVP is PVP-K60 or PVP-K90.

[0160] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising PEG. In some embodiments, the PEG is PEG-200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is a low molecular weight PEG. In some embodiments, the low molecular weight PEG is PEG-200, PEG-300, PEG-400, PEG-600, or PEG-1500. In some embodiments, the PEG is PEG-1500. In some embodiments, PEG-1500 comprises PEG-300 or PEG1540. In some embodiments, the PEG is a medium molecular weight PEG. In some embodiments, the medium molecular weight PEG is PEG-1000, PEG- 1540, PEG-2000. In some embodiments, the PEG is a high molecular weight PEG. In some embodiments, the high molecular weight PEG is PEG-3100, PEG-3400, PEG-8300, PEG- 8600, PEG-11000, PEG-13000, or PEG-20000.

[0161] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising a buffer. In some embodiments, the buffer has a pH from about 4.0 to about 8.0, for example from about 5.0 to about 7.0, e.g., from about 5.8 to about 6.2. Examples of buffers include, but are not limited to, acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In one embodiment herein, the buffer is a histidine buffer. In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0162] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising a phosphate-buffered saline (PBS) buffer. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline.

[0163] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising a Tris buffer. In some embodiments, the Tris is Tris - EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris is Tris-Boric Acid-EDTA (TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE). In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris has a pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3.

[0164] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising an acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In someembodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0165] The reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PEG is PEG- 200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG- 12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0166] The reconstituted formulation is obtained by reconstituting a lyophilized formulation comprising an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PVP is PVP- K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0167] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an oligosaccharide. The reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is PBS. In some embodiments, the lyoprotectant comprises anoligosaccharide and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is an acetate buffer. In some embodiments, the oligosaccharide is sucrose, maltose, trehalose, lactose, glucose, or any combination thereof. In some embodiments, the oligosaccharide is sucrose. In some embodiments, the oligosaccharide is maltose. In some embodiments, the oligosaccharide is trehalose. In some embodiments, the oligosaccharide is lactose. In some embodiments, the oligosaccharide is glucose.

[0168] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 0%-40%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-10%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 10%-l 5%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 15%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 20%-25%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 25%-30%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 30%-35%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 35%-40%. In some embodiments, the concentration of sucrose is 5%. In some embodiments, the concentration of sucrose is 10%. In some embodiments, the concentration of sucrose is 15%. In some embodiments, the concentration of sucrose is 20%. In some embodiments, the concentration of sucrose is 25%. In some embodiments, the concentration of sucrose is 30%. In some embodiments, the concentration of sucrose is 35%. In some embodiments, the concentration of sucrose is 40%.

[0169] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 0%-40%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-10%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 10%-l 5%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 15%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 20%-25%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 25%-30%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 30%-35%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 35%-40%. In some embodiments, the concentration of maltose is 5%. In some embodiments, the concentration of maltose is 10%. In some embodiments, the concentration of maltose is 15%. In some embodiments, the concentration of maltose is 20%. In some embodiments, the concentration of maltose is 25%. In some embodiments, the concentration of maltose is 30%. In some embodiments, the concentration of maltose is 35%. In some embodiments, the concentration of maltose is 40%.

[0170] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 0%-40%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 5%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucoseat a concentration of 5%-10%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 10%- 15%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 15%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 20%-25%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 25%-30%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 30%-35%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 35%-40%. In some embodiments, the concentration of trehalose or lactose or glucose is 5%. In some embodiments, the concentration of trehalose or lactose or glucose is 10%. In some embodiments, the concentration of trehalose or lactose or glucose is 15%. In some embodiments, the concentration of trehalose or lactose or glucose is 20%. In some embodiments, the concentration of trehalose or lactose or glucose is 25%. In some embodiments, the concentration of trehalose or lactose or glucose is 30%. In some embodiments, the concentration of trehalose or lactose or glucose is 35%. In some embodiments, the concentration of trehalose or lactose or glucose is 40%.

[0171] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an amino acid. The reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an amino acid. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is PBS. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is an acetate buffer. In some embodiments, the amino acid is lysine, arginine, leucine, glutamic acid, or any combination thereof. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is arginine. In someembodiments, the amino acid is leucine. In some embodiments, the amino acid is glutamic acid.

[0172] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 0%-40%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-10%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 10%-l 5%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 15%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 20%-25%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 25%-30%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 30%-35%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 35%-40%. In some embodiments, the concentration of lysine is 5%. In some embodiments, the concentration of lysine is 10%. In some embodiments, the concentration of lysine is 15%. In some embodiments, the concentration of lysine is 20%. In some embodiments, the concentration of lysine is 25%. In some embodiments, the concentration of lysine is 30%. In some embodiments, the concentration of lysine is 35%. In some embodiments, the concentration of lysine is 40%.

[0173] In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 0%-40%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at aconcentration of 5%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 5%-10%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 10%- 15%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 15%-20%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 20%-25%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 25%-30%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 30%-35%. In some embodiments, the reconstituted formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 35%-40%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 5%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 10%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 15%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 20%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 25%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 30%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 35%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 40%.

[0174] In some embodiments, the reconstituted formulation comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA is a single-stranded DNA (ssDNA), doublestranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. In some embodiments, the DNA is a plasmid, oligonucleotide, antisense oligonucleotide (ASO), DNA aptamer, or DNAzyme. In some embodiments, the DNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15-20 nucleotides in length. In someembodiments, the ASO binds a complementary RNA target. In some embodiments, the DNA is circular. In some embodiments, the DNA is linear. In some embodiments, the DNA is copy DNA or complementary DNA (cDNA). In some embodiments, the DNA is a synthetic DNA. In some embodiments, the DNA is a PCR amplicon. In some embodiments, the DNA is synthesized by endonuclease-mediated assembly, BioBricks, Golden Gate cloning, or Gibson assembly. In some embodiments, the DNA is a naturally occurring DNA. In some embodiments, the DNA is extracted from a cell or tissue. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 30 pg / ml. Insome embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 100 pg / ml.

[0175] In some embodiments, the reconstituted formulation comprises a ribonucleic acid (RNA). In some embodiments, the RNA is a circular RNA or a linear RNA. In some embodiments, the RNA is linear RNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is a messenger RNA (mRNA). In some embodiments, the RNA is an engineered RNA. In some embodiments, the RNA is a naturally occurring RNA. In some embodiments, the RNA is a non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. In some embodiments, the RNA is non-coding RNAs (ncRNA) that is not translated into a protein, e.g., small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), CRISPR RNA (crRNA), tracrRNA, guide RNA gRNA), and extracellular RNA (exRNA). In some embodiments, the RNA is a micro RNA (miRNA). In some embodiments, the RNA is a small inhibitory RNA (siRNA). In some embodiments, the RNA is a CRISPR-associated RNA, e.g., gRNA, crRNA, tracrRNA. In some embodiments, the RNA is a guide RNA (gRNA). In some embodiments, the RNA is small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). In some embodiments, the RNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15- 20 nucleotides in length. In some embodiments, the ASO binds a complementary RNA target. The RNA can be double-stranded RNA or single-stranded RNA. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in thereconstituted NA-LNP formulation is between 5 gg / ml and 50 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 gg / ml and 100 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 5 gg / ml and 10 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 10 gg / ml and 20 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 20 gg / ml and 30 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 30 gg / ml and 40 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 40 gg / ml and 50 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 50 gg / ml and 60 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 60 gg / ml and 70 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 70 gg / ml and 80 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 80 gg / ml and 90 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is between 90 gg / ml and 100 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 5 gg / ml, 10 gg / ml, 20 gg / ml, 30 gg / ml, 40 gg / ml 50 gg / ml, 60 gg / ml, 70 gg / ml, 80 gg / ml, 90 gg / ml or 100 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 5 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 10 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 20 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 30 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 40 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 50 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 60 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 70 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 80 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 90 gg / ml. In some embodiments, the concentration of the nucleic acid in the reconstituted NA-LNP formulation is 100 gg / ml.

[0176] As used herein, “LNPs” refer to “lipid nanoparticles,” which are nanoparticles comprising lipids. In some embodiments, the LNP is spherical. In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm and about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm to about 75 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 75 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 100 nm to about 125 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 90 nm to about 110 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 125 nm to about 150 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 150 nm to about 200 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 200 nm to about 250 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 250 nm to about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 1000 nm. In some embodiments, the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 50 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm to about 200 nm. In some embodiments, the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 200 nm to about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 300 nm to about 400 nm. In some embodiments, the diameter of the NA-LNP particlein the aqueous NA-LNP formulation is about 400 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 500 nm to about 600 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 600 nm to about 700 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 700 nm to about 800 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 800 nm to about 900 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 900 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm, 25 nm, 50 nm, 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0177] As used herein, “LNPs” refer to “lipid nanoparticles,” which are nanoparticles comprising lipids. In some embodiments, the LNP is spherical. In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 50 nm and about 300 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 50 nm to about 75 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 75 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 100 nm to about 125 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 90 nm to about 110 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 125 nm to about 150 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 150 nm to about 200 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 200 nm to about 250 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is between about 250 nm to about 300 nm. In some embodiments, the lyophilized formulationcomprises NA-LNP particles, and the diameter of the NA-LNP particle is about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 50 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA- LNP particles, and the diameter of the NA-LNP particle is about 1 nm to about 100 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 100 nm to about 200 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 200 nm to about 300 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 300 nm to about 400 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 400 nm to about 500 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 500 nm to about 600 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 600 nm to about 700 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 700 nm to about 800 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 800 nm to about 900 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 900 nm to about 1000 nm. In some embodiments, the lyophilized formulation comprises NA-LNP particles, and the diameter of the NA-LNP particle is about 10 nm, 25 nm, 50 nm, 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0178] LNP structure is defined by its core, inner shell, and outer shell. The nucleic acid payload is encapsulated in a liquid crystalline phase in the core. The inner and outer shell are comprised of lipids. In some embodiments, the nanoparticles comprise at least one lipid. In some embodiments, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises 1,2-di stearoyl -sn- glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k. In some embodiments, the LNP comprises cholesterol. In some embodiments, the LNP comprises an ionizable lipid. In some embodiments, the LNP comprises a PEGylated lipid. In some embodiments, the LNP comprises cholesterol and an ionizable lipid. An ionizable lipid is a lipid that is positive at acidic pH and neutral at basic pH. Examples of ionizable lipids include ALC-0315, C12-200, cKK-E12, and DLin-MC3-DMA. In some embodiments, the LNP comprises cholesterol, an ionizable lipid, and DSPC. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the nucleic acid. In one embodiment, the at least one cationic lipid comprises l,2-di-0-octadecenyl-3- trimethylammonium propane (DOTMA) or analogs or derivatives thereof and / or 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof. In one embodiment, the nanoparticles comprise at least one helper lipid. Helper lipids are a class of lipid molecules that increase particle stability and fluidity of lipid nanoparticles (LNP). The helper lipid may be a neutral or an anionic lipid. Examples of helper lipids include DSPC, cholesterol, and PEGylated lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In some embodiments, the cationic lipid and / or the helper lipid is a bilayer forming lipid. In one embodiment, the at least one helper lipid comprises 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and / or 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC) or analogs or derivatives thereof. In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1 :2, 4: 1 to 2:3, 7:3 to 1 : 1, or 2: 1 to 1 : 1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molaramount of the cationic lipid multiplied by the number of positive charges in the cationic lipid. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm.

[0179] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation and the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 10%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 15%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 20%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and 25%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 30%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 15% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 30% and 40% weight per volume (w / v). In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the concentration of the lyoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, or 90% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 5% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 10% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 45% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 50% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 55% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 60% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 65% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 70% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 75% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 80% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 90% weight per volume (w / v). In some embodiments, the lyoprotectant is PVP. In other embodiments, the lyoprotectant is PEG. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG- 20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, thelyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0180] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising PBS. In some embodiments, the PBS in the aqueous NA-LNP formulation is lx PBS, 5x PBS or lOx PBS. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is PBS.

[0181] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising a phosphate buffer. In some embodiments, the phosphate buffer has a pH of about 7.0 to about 9.0. In some embodiments, the phosphatebuffer has a pH of about 7.0 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.8. In some embodiments, the phosphate buffer has a pH of about 8.0 to about 8.5. In some embodiments, the phosphate buffer has a pH of about 8.5 to about 9.0. In some embodiments, the phosphate buffer has a pH of about 7.4. In some embodiments, the phosphate buffer has a pH of about 7.5. In some embodiments, the phosphate buffer has a pH of about 7.6. In some embodiments, the phosphate buffer has a pH of about 8.0. In some embodiments, the phosphate buffer has a pH of about 8.3. In some embodiments, the concentration of the phosphate buffer in the reconstituted formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 5 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 10 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 20 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 25 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 50 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 100 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 150 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 200 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 250 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 300 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 400 mM. In some embodiments, the concentration of phosphate buffer in the reconstituted NA-LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer.

[0182] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising Tris. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 150 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 200 mM and 300 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 350 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 300 mM and 400 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 350 mM and 450 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 400 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 50 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 20 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 45 mM and 55 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 70 mM and 80 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 80 mM and 90 mM. Insome embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA- LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 75 mM and 125 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, or IM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris is Tris-Boric Acid-EDTA (TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE) In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris has a pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP- K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP- K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG- 3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is Tris.

[0183] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation comprising acetate buffer. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 500 mM. Theconcentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 150 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 150 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 250 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 250 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 350 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 350 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 450 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 450 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 10 mM and 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation isbetween 70 mM and 80 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 80 mM and 90 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 110 mM and 120 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 120 mM and 130 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 130 mM and 140 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA- LNP formulation is between 140 mM and 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 5 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 200 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 250 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 300 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 400 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetatetrihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In some embodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0184] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 2.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 2.0 and about pH 3.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 3.0 and about pH 4.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.0 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 7.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 9.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 9.0 and about pH 10.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 10.0 and about pH 11.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 11.0 and about pH 12.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation isbetween about pH 12.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 4.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.5 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.0 and about pH 5.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.5 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 6.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.3 and about pH 6.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.4 and about pH 6.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.5 and about pH 7.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.7 and about pH 7.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.2 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.4 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.8 and about pH 8.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.5 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 8.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.2, pH 8.4, or pH 8.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0. In some embodiments, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is asodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0185] In some embodiments, the reconstituted formulation is obtained by reconstituting a lyophilized formulation. In some embodiments, the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNPformulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA is mRNA.IV. Aqueous formulations

[0186] In one embodiment, the invention provides an aqueous formulation comprising a NA- LNP particle. The NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle. The formulation further comprises an excipient comprising a lyoprotectant and a buffer. The lyoprotectant is polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG).

[0187] In some embodiments, an aqueous formulation is a fresh formulation as described in Examples 1-3. In some embodiments, the fresh formulation is a freshly prepared formulation. In some embodiments, the fresh formulation has not been previously lyophilized. In some embodiments, the fresh formulation has not been previously reconstituted. In some embodiments, the fresh formulation has not been previously frozen. In some embodiments, the fresh formulation has not been previously thawed. In some embodiments, the fresh formulation has not been previously lyophilized, reconstituted, or frozen or thawed.

[0188] In some embodiments, the aqueous formulation comprises PVP. PVP is also known as polyvidone or povidone. PVP is a polymer molecule which comprises the monomer N- vinylpyrrolidone. In some embodiments, the PVP is entirely or substantially composed of N- vinylpyrrolidone. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP- K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments the PVP is low molecular weight PVP. In some embodiments, the low molecular weight PVP is PVP-K12, PVP -KI 5, or PVP-K17. In some embodiments, the PVP is PVP-K12. In some embodiments the PVP is medium molecular weight PVP. In some embodiments, the medium molecular weight PVP is PVP-K25 or PVP-K30. In some embodiments the PVP is high molecular weight PVP. In some embodiments, the high molecular weight PVP is PVP-K60 or PVP-K90.

[0189] In some embodiments, the aqueous formulation comprises PEG. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is a low molecular weight PEG. In some embodiments, the low molecular weight PEG is PEG-200, PEG-300, PEG-400, PEG-600, or PEG-1500. In some embodiments, the PEG is PEG-1500. In some embodiments, PEG-1500 comprises PEG-300 or PEG1540. In some embodiments, the PEG is a medium molecular weight PEG. In some embodiments, the medium molecular weight PEG is PEG- 1000, PEG- 1540, PEG-2000. In some embodiments, the PEG is a high molecular weight PEG. In some embodiments, the high molecular weight PEG is PEG-3100, PEG-3400, PEG-8300, PEG-8600, PEG-11000, PEG-13000, or PEG-20000.

[0190] In some embodiments, the aqueous formulation comprises a buffer. As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In some embodiments, the buffer has a pH from about 4.0 to about 8.0, for example from about 5.0 to about 7.0, e.g. from about 5.8 to about 6.2. Examples of buffers include, but are not limited to, acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In one embodiment herein, the buffer is a histidine buffer. In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0191] In some embodiments, the aqueous formulation comprises a phosphate-buffered saline (PBS) buffer. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline.

[0192] In some embodiments, the aqueous formulation comprises a Tris buffer. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate- EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris isTris-Boric Acid-EDTA (TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE). In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris has a pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3.

[0193] In some embodiments, the aqueous formulation comprises a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In some embodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0194] In some embodiments, the aqueous formulation comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0195] In some embodiments, the aqueous formulation comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is an acetate buffer. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PBS is 0.5x, lx, 2x, 3x, 4x, 5x, lOx, 20x, or 50x. In some embodiments, the Tris is TE, TAE, Tris-HCl, TBE, TPE, or TG. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer.

[0196] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises an oligosaccharide. The aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is PBS. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is an acetate buffer. In some embodiments, the oligosaccharide is sucrose, maltose, trehalose, lactose, glucose, or any combination thereof. In some embodiments, the oligosaccharide is sucrose. In some embodiments, the oligosaccharide is maltose. In some embodiments, the oligosaccharide is trehalose. In some embodiments, the oligosaccharide is lactose. In some embodiments, the oligosaccharide is glucose.

[0197] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises sucrose. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 0%-40%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 5%-l 0%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 10%- 15%. In someembodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 15%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 20%-25%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 25%-30%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 30%-35%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises sucrose at a concentration of 35%-40%. In some embodiments, the concentration of sucrose is 5%. In some embodiments, the concentration of sucrose is 10%. In some embodiments, the concentration of sucrose is 15%. In some embodiments, the concentration of sucrose is 20%. In some embodiments, the concentration of sucrose is 25%. In some embodiments, the concentration of sucrose is 30%. In some embodiments, the concentration of sucrose is 35%. In some embodiments, the concentration of sucrose is 40%.

[0198] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises maltose. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 0%-40%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 5%-l 0%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises maltose at a concentration of 10%-l 5%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 15%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 20%-25%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 25%-30%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of 30%-35%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises maltose at a concentration of35%-40%. In some embodiments, the concentration of maltose is 5%. In some embodiments, the concentration of maltose is 10%. In some embodiments, the concentration of maltose is 15%. In some embodiments, the concentration of maltose is 20%. In some embodiments, the concentration of maltose is 25%. In some embodiments, the concentration of maltose is 30%. In some embodiments, the concentration of maltose is 35%. In some embodiments, the concentration of maltose is 40%.

[0199] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 0%-40%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 5%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 5%-l 0%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 10%-l 5%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 15%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 20%-25%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 25%-30%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 30%-35%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises trehalose or lactose or glucose at a concentration of 35%-40%. In some embodiments, the concentration of trehalose or lactose or glucose is 5%. In some embodiments, the concentration of trehalose or lactose or glucose is 10%. In some embodiments, the concentration of trehalose or lactose or glucose is 15%. In some embodiments, the concentration of trehalose or lactose or glucose is 20%. In some embodiments, the concentration of trehalose or lactose or glucose is 25%. In some embodiments, the concentration of trehalose or lactose or glucose is 30%. In some embodiments, theconcentration of trehalose or lactose or glucose is 35%. In some embodiments, the concentration of trehalose or lactose or glucose is 40%.

[0200] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises an amino acid. The aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises an excipient comprising a lyoprotectant and a buffer. In some embodiments, the lyoprotectant comprises an amino acid. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is PBS. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is an acetate buffer. In some embodiments, the amino acid is lysine, arginine, leucine, glutamic acid, or any combination thereof. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is arginine. In some embodiments, the amino acid is leucine. In some embodiments, the amino acid is glutamic acid.

[0201] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises lysine. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 0%-40%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 5%-l 0%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises lysine at a concentration of 10%- 15%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 15%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 20%-25%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 25%-30%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of 30%-35%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises lysine at a concentration of35%-40%. In some embodiments, the concentration of lysine is 5%. In some embodiments, the concentration of lysine is 10%. In some embodiments, the concentration of lysine is 15%. In some embodiments, the concentration of lysine is 20%. In some embodiments, the concentration of lysine is 25%. In some embodiments, the concentration of lysine is 30%. In some embodiments, the concentration of lysine is 35%. In some embodiments, the concentration of lysine is 40%.

[0202] In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 0%-40%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 5%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)- lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 5%-10%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 10%-l 5%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 15%-20%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 20%-25%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 25%-30%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 30%-35%. In some embodiments, the aqueous formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises arginine or leucine or glutamic acid at a concentration of 35%-40%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 5%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 10%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 15%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 20%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 25%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 30%. In someembodiments, the concentration of arginine or leucine or glutamic acid is 35%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 40%.

[0203] In some embodiments, the aqueous formulation comprises a deoxyribonucleic acid (DNA). In some embodiments, the DNA is a single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA / DNA hybrids. In some embodiments, the DNA is a plasmid, oligonucleotide, antisense oligonucleotide (ASO), DNA aptamer, or DNAzyme. In some embodiments, the DNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15-20 nucleotides in length. In some embodiments, the ASO binds a complementary RNA target. In some embodiments, the DNA is circular. In some embodiments, the DNA is linear. In some embodiments, the DNA is copy DNA or complementary DNA (cDNA). In some embodiments, the DNA is a synthetic DNA. In some embodiments, the DNA is a PCR amplicon. In some embodiments, the DNA is synthesized by endonuclease-mediated assembly, BioBricks, Golden Gate cloning, or Gibson assembly. In some embodiments, the DNA is a naturally occurring DNA. In some embodiments, the DNA is extracted from a cell or tissue. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acidin the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 qg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml.

[0204] In some embodiments, the aqueous formulation comprises a ribonucleic acid (RNA). In some embodiments, the RNA is a circular RNA or a linear RNA. In some embodiments, the RNA is linear RNA. In some embodiments, the RNA is circular RNA. In some embodiments, the RNA is a messenger RNA (mRNA). In some embodiments, the RNA is an engineered RNA. In some embodiments, the RNA is a naturally occurring RNA. In some embodiments, the RNA is a non-capped mRNA, a non-polyadenylated mRNA, and a non-spliced mRNA. In some embodiments, the RNA is non-coding RNAs (ncRNA) that is not translated into a protein, e.g., small non-coding RNAs such as microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), CRISPR RNA (crRNA), tracrRNA, guide RNA gRNA), and extracellular RNA (exRNA). In some embodiments, the RNA is a micro RNA (miRNA). In some embodiments, the RNA is a small inhibitory RNA (siRNA). In some embodiments, the RNA is a CRISPR-associated RNA, e.g., gRNA, crRNA, tracrRNA. Insome embodiments, the RNA is a guide RNA (gRNA). In some embodiments, the RNA is small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA greater than 200 nucleic acid bases in length). Examples of small RNAs include miRNA, siRNA, snoRNAs, piRNA, tRNA-derived small RNA (tsRNA), and small rDNA-derived RNA (srRNA). In some embodiments, the RNA is an antisense oligonucleotide (ASO), also known as an antisense agent. In some embodiments, the ASO is 15-35 nucleotides in length. In some embodiments, the ASO is 15-25 nucleotides in length. In some embodiments, the ASO is 15- 20 nucleotides in length. In some embodiments, the ASO binds a complementary RNA target. The RNA can be double-stranded RNA or single-stranded RNA. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml 50 pg / ml, 60 pg / ml, 70 pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In someembodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml.

[0205] As used herein, “LNPs” refer to nanoparticles comprising lipids. In some embodiments, the LNP is spherical. In some embodiments, diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm and about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 50 nm to about 75 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 75 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 100 nm to about 125 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 90 nm to about 110 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 125 nm to about 150 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 150 nm to about 200 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 200 nm to about 250 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between about 250 nm to about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA- LNP formulation is about 1 nm to about 50 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm to about 500 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 1 nm to about 100 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm to about 200 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 200 nm to about 300 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 300 nm to about 400 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 400 nm to about 500 nm. In some embodiments, the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 500 nm to about 600 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 600 nm to about 700 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 700 nm to about 800 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 800 nm to about 900 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 900 nm to about 1000 nm. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 10 nm, 25 nm, 50 nm, 75 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 450 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, or about 1000 nm.

[0206] LNP structure is defined by its core, inner shell, and outer shell. The nucleic acid payload is encapsulated in a liquid crystalline phase in the core. The inner and outer shell are comprised of lipids. In some embodiments, the nanoparticles comprise at least one lipid. In some embodiments, the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof. In some embodiments, the ionizable lipid comprises SM-102. In some embodiments, the phospholipid comprises 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC). In some embodiments, the PEGylated lipid comprises DMG-PEG2k. In some embodiments, the LNP comprises cholesterol. In some embodiments, the LNP comprises an ionizable lipid. In some embodiments, the LNP comprises a PEGylated lipid. In some embodiments, the LNP comprises cholesterol and an ionizable lipid. An ionizable lipid is a lipid that is positive at acidic pH and neutral at basic pH. Examples of ionizable lipids include ALC-0315, C12-200, cKK-E12, and DLin-MC3-DMA.In some embodiments, the LNP comprises cholesterol, an ionizable lipid, and DSPC. In one embodiment, the nanoparticles comprise at least one cationic lipid. The cationic lipid can be monocationic or polycationic. Any cationic amphiphilic molecule, e.g., a molecule which comprises at least one hydrophilic and lipophilic moiety is a cationic lipid within the meaning of the present invention. In one embodiment, the positive charges are contributed by the at least one cationic lipid and the negative charges are contributed by the nucleic acid. In one embodiment, the at least one cationic lipid comprises l,2-di-0-octadecenyl-3- trimethylammonium propane (DOTMA) or analogs or derivatives thereof and / or 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP) or analogs or derivatives thereof. In one embodiment, the nanoparticles comprise at least one helper lipid. Helper lipids are a class of lipid molecules that increase particle stability and fluidity of lipid nanoparticles (LNP). The helper lipid may be a neutral or an anionic lipid. Examples of helper lipids include DSPC, cholesterol, and PEGylated lipid. The helper lipid may be a natural lipid, such as a phospholipid or an analogue of a natural lipid, or a fully synthetic lipid, or lipid-like molecule, with no similarities with natural lipids. In some embodiments, the cationic lipid and / or the helper lipid is a bilayer forming lipid. In one embodiment, the at least one helper lipid comprises 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) or analogs or derivatives thereof, cholesterol (Choi) or analogs or derivatives thereof and / or 1,2- dioleoyl-sn-glycero-3 -phosphocholine (DOPC) or analogs or derivatives thereof. In one embodiment, the molar ratio of the at least one cationic lipid to the at least one helper lipid is from 10:0 to 3:7, preferably 9: 1 to 3:7, 4: 1 to 1 :2, 4: 1 to 2:3, 7:3 to 1 : 1, or 2: 1 to 1 : 1. In one embodiment, in this ratio, the molar amount of the cationic lipid results from the molar amount of the cationic lipid multiplied by the number of positive charges in the cationic lipid. In some embodiments, the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm.

[0207] The aqueous NA-LNP formulation comprises a lyoprotectant. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 10%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 15%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 20%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and25%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 30%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40%. In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 15% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 10% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 15% and 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 25% and 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 35% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 20% and 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 30% and 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, or 90% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 5% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 10% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 20% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 25% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 30% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 35% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 40% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 45% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 50% weight per volume(w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 55% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 60% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 65% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 70% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 75% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 80% weight per volume (w / v). In some embodiments, the concentration of the lyoprotectant in the aqueous NA-LNP formulation is about 90% weight per volume (w / v). In some embodiments, the lyoprotectant is PVP. In other embodiments, the lyoprotectant is PEG. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG- 20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0208] In some embodiments, the aqueous NA-LNP formulation comprises PBS. In some embodiments, the PBS in the aqueous NA-LNP formulation is lx PBS, 5x PBS or lOx PBS. In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 5x PBS. In someembodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, the PBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP- K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is PBS.

[0209] In some embodiments, the aqueous NA-LNP formulation comprises a phosphate buffer. In some embodiments, the phosphate buffer has a pH of about 7.0 to about 9.0. In some embodiments, the phosphate buffer has a pH of about 7.0 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.6. In some embodiments, the phosphate buffer has a pH of about 7.4 to about 7.8. In some embodiments, the phosphate buffer has a pH of about 8.0 to about 8.5. In some embodiments, the phosphate buffer has a pH of about 8.5 to about 9.0. In some embodiments, the phosphate buffer has a pH of about 7.4. In some embodiments, the phosphate buffer has a pH of about 7.5. In some embodiments, the phosphate buffer has a pH of about 7.6. In some embodiments, the phosphate buffer has a pH of about 8.0. In some embodiments, the phosphate buffer has a pH of about 8.3. In some embodiments, the concentration of the phosphate buffer in the aqueous formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 5 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is100 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA- LNP formulation is 150 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 200 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 250 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 300 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA-LNP formulation is 400 mM. In some embodiments, the concentration of phosphate buffer in the aqueous NA- LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP- K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP- K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG- 3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG- 1500. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer.

[0210] In some embodiments, the aqueous NA-LNP formulation comprises Tris. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 100 mM and 150 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 200 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 150 mM and 250 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 200 mM and 300 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 250 mM and 350 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 300 mM and 400 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 350 mM and 450 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 400 mM and 500 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNPformulation is between 50 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 5 mM and 20 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 45 mM and 55 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 70 mM and 80 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 80 mM and 90 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is between 75 mM and 125 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 250 mM, 500 mM, or IM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of Tris in the aqueous NA- LNP formulation is 50 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of Tris in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris-HCl. In some embodiments, the Tris is Tris-Boric Acid-EDTA (TBE). In some embodiments, the Tris is Tris-Phosphate-EDTA (TPE) In some embodiments, the Tris is Tris-Glycine (TG). In some embodiments, the Tris has a pH of about 7.0 to about 9.0. In some embodiments, the Tris has a pH of about 7.0 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.6. In some embodiments, the Tris has a pH of about 7.4 to about 7.8. In some embodiments, the Tris has a pH of about 8.0 to about 8.5. In some embodiments, the Tris has a pH of about 8.5 to about 9.0. In some embodiments, the Tris hasa pH of about 7.4. In some embodiments, the Tris has a pH of about 7.5. In some embodiments, the Tris has a pH of about 7.6. In some embodiments, the Tris has a pH of about 8.0. In some embodiments, the Tris has a pH of about 8.3. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is Tris.

[0211] In some embodiments, the aqueous NA-LNP formulation comprises an acetate buffer. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 150 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 150 mM and 200 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 200 mM and 250 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 250 mM and 300 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 300 mM and 350 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 350 mM and 400 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 450 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 450 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 400 mM and 500 mM. The concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 50 mM. In someembodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 10 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 10 mM and 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 20 mM and 30 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 30 mM and 40 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 40 mM and 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 50 mM and 60 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 60 mM and 70 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 70 mM and 80 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 80 mM and 90 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 100 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 90 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 100 mM and 110 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA- LNP formulation is between 110 mM and 120 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 120 mM and 130 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 130 mM and 140 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is between 140 mM and 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 5 mM, 10 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 400 mM, 500 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA- LNP formulation is 5 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 10 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 20 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 25 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 50 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 100 mM. In some embodiments, the concentration of acetate buffer in theaqueous NA-LNP formulation is 150 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 200 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 250 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 300 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 400 mM. In some embodiments, the concentration of acetate buffer in the aqueous NA-LNP formulation is 500 mM. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments, the PVP is PVP-K12. In some embodiments, the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is PEG-1500. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the acetate buffer is sodium acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer in combination with acetic acid. In some embodiments, the acetate buffer is sodium acetate trihydrate buffer. In some embodiments, the acetate buffer has a pH of about 3.6 to about 7.0. In some embodiments, the acetate buffer has a pH of about 3.6 to about 4.0. In some embodiments, the acetate buffer has a pH of about 4.0 to about 5.0. In some embodiments, the acetate buffer has a pH of about 4.5 to about 5.5. In some embodiments, the acetate buffer has a pH of about 6.0 to about 6.5. In some embodiments, the acetate buffer has a pH of about 6.5 to about 7.0. In some embodiments, the acetate buffer has a pH of about 5.0.

[0212] In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 1.0 and about pH 2.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 2.0 and about pH 3.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 3.0 and about pH 4.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueousNA-LNP formulation is between about pH 5.0 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 7.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.0 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 9.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 9.0 and about pH 10.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 10.0 and about pH 11.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 11.0 and about pH 12.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 12.0 and about pH 13.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.0 and about pH 4.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 4.5 and about pH 5.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.0 and about pH 5.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 5.5 and about pH 6.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.0 and about pH 6.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.3 and about pH 6.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.4 and about pH 6.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.5 and about pH 7.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 6.7 and about pH 7.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.2 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.4 and about pH 7.8. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.8 and about pH 8.2. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 7.5 and about pH 8.0. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is between about pH 8.0 and about pH 8.5. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0, pH 5.0, pH 6.0, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.2, pH 8.4, or pH 8.6. In some embodiments, the pH of the buffer in the aqueous NA-LNP formulation is or is about pH 4.0. In some embodiments, the buffer is phosphate-buffered saline (PBS), aphosphate buffer, a Tris buffer or an acetate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is PBS. In some embodiments, the lyoprotectant is PVP and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PVP and the buffer is Tris. In some embodiments, the lyoprotectant is PVP and the buffer is acetate. In some embodiments, the lyoprotectant is PVP, and the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is PBS. In some embodiments, the lyoprotectant is PEG and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant is PEG and the buffer is Tris. In some embodiments, the lyoprotectant is PEG and the buffer is acetate. In some embodiments, the acetate buffer is a sodium acetate trihydrate buffer. In some embodiments, the lyoprotectant is PEG, and the acetate buffer is a sodium acetate trihydrate buffer.

[0213] The aqueous NA-LNP formulation comprises a nucleic acid. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA- LNP formulation is between 5 pg / ml and 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 10 pg / ml and 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 20 pg / ml and 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 30 pg / ml and 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 40 pg / ml and 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 50 pg / ml and 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 60 pg / ml and 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 70 pg / ml and 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 80 pg / ml and 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 90 pg / ml and 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70pg / ml, 80 pg / ml, 90 pg / ml or 100 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 5 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 20 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 30 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 40 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 50 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 60 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 70 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 80 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 90 pg / ml. In some embodiments, the concentration of the nucleic acid in the aqueous NA-LNP formulation is 100 pg / ml. In some embodiments, the nucleic acid comprises DNA. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA is mRNA.

[0214] In some embodiments, the aqueous formulation when lyophilized and reconstituted results in a diameter of the NA-LNP particle in the reconstituted formulation that is no more than 1% larger, 2% larger, 3% larger, 4% larger, 5% larger, 10% larger, 12.5% larger, 15% larger, 20% larger, 25% larger, 30% larger, 35% larger, 40% larger, or 50% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 1% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 2% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 3% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA- LNP particle in the reconstituted formulation is no more than 4% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 5% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is nomore than 10% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 12.5% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 15% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA- LNP particle in the reconstituted formulation is no more than 20% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 25% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 30% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 35% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 40% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA- LNP particle in the reconstituted formulation is no more than 45% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the diameter of the NA-LNP particle in the reconstituted formulation is no more than 50% larger than the diameter of the NA-LNP particle in the aqueous NA-LNP formulation.

[0215] In some embodiments, the aqueous formulation when lyophilized and reconstituted results in a percent encapsulation efficiency (%EE) of the nucleic acid in the NA-LNP particle in the reconstituted formulation that is no more than 1% lower, 2% lower, 3% lower, 4% lower, 5% lower, 10% lower, 12.5% lower, 15% lower, 20% lower, 25% lower, 30% lower, 35% lower, 40% lower, 45% lower, or 50% lower than the percent encapsulation efficiency (%EE) of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 1% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 2% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNPparticle in the reconstituted formulation is no more than 3% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 4% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 5% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 12.5% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 15% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 20% lower than the %EE of the of the nucleic acid in the NA- LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 25% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA- LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 30% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 35% lower than the %EE of the of the nucleic acid in the NA- LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 40% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA- LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 45% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the %EE of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 50% lower than the %EE of the of the nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the percent encapsulation efficiency (%EE) is calculated asSoluble nucleic acid concentration%EE = (1 - — - - - — - - -;- ) x 100%Total nucleic acid concentration

[0216] In some embodiments, the aqueous formulation when lyophilized and reconstituted results in an amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 1% lower, 2% lower, 3% lower, 4% lower, 5% lower, 10% lower, 15% lower, 20% lower, 25% lower, or 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 1% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 2% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 3% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 4% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 5% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 15% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 20% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 25% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation. In some embodiments, the amount of intact nucleic acid in the NA-LNP particlein the reconstituted formulation is no more than 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the aqueous NA-LNP formulation.V. Method of making lyophilized formulations

[0217] Also provided are methods of making a lyophilized formulation comprising a NA- LNP particle. The NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle. The method comprises: i) generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii) adding an excipient comprising a lyoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation, wherein the lyoprotectant is polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG); and iii) lyophilizing the aqueous NA-LNP formulation to produce the lyophilized formulation.

[0218] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyoprotectant is PVP. PVP is also known as polyvidone or povidone. PVP is a polymer molecule which comprises the monomer N-vinylpyrrolidone. In some embodiments, the PVP is entirely or substantially composed of N-vinylpyrrolidone. In some embodiments, the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, or PVP-K90. In some embodiments the PVP is low molecular weight PVP. In some embodiments, the low molecular weight PVP is PVP-K12, PVP-K15, or PVP-K17. In some embodiments, the PVP is PVP-K12. In some embodiments the PVP is medium molecular weight PVP. In some embodiments, the medium molecular weight PVP is PVP-K25 or PVP- K30. In some embodiments the PVP is high molecular weight PVP. In some embodiments, the high molecular weight PVP is PVP-K60 or PVP-K90.

[0219] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle comprises PEG. In some embodiments, the PEG is PEG-200, PEG-400, PEG- 600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000. In some embodiments, the PEG is a low molecular weight PEG. In some embodiments, the low molecular weight PEG is PEG-200, PEG-300, PEG-400, PEG-600, or PEG-1500. In some embodiments, the PEG is PEG-1500. In some embodiments, PEG-1500 comprises PEG-300 or PEG1540. In some embodiments, the PEG is a medium molecular weight PEG.In some embodiments, the medium molecular weight PEG is PEG-1000, PEG-1540, PEG- 2000. In some embodiments, the PEG is a high molecular weight PEG. In some embodiments, the high molecular weight PEG is PEG-3100, PEG-3400, PEG-8300, PEG- 8600, PEG-11000, PEG-13000, or PEG-20000.

[0220] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyoprotectant comprises an oligosaccharide. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is PBS. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a phosphate buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an oligosaccharide and the buffer is an acetate buffer. In some embodiments, the oligosaccharide is sucrose, maltose, trehalose, lactose, glucose, or any combination thereof. In some embodiments, the oligosaccharide is sucrose. In some embodiments, the oligosaccharide is maltose. In some embodiments, the oligosaccharide is trehalose. In some embodiments, the oligosaccharide is lactose. In some embodiments, the oligosaccharide is glucose.

[0221] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding sucrose as the lyoprotectant in step ii). In some embodiments, sucrose is added at a concentration of 0%-40%. In some embodiments, sucrose is added at a concentration of 5%-20%. In some embodiments, sucrose is added at a concentration of 5%- 10%. In some embodiments, sucrose is added at a concentration of 10%- 15%. In some embodiments, sucrose is added at a concentration of 15%-20%. In some embodiments, sucrose is added at a concentration of 20%-25%. In some embodiments, sucrose is added at a concentration of 25%-30%. In some embodiments, sucrose is added at a concentration of 30%-35%. In some embodiments, sucrose is added at a concentration of 35%-40%. In some embodiments, the concentration of sucrose is 5%. In some embodiments, the concentration of sucrose is 10%. In some embodiments, the concentration of sucrose is 15%. In some embodiments, the concentration of sucrose is 20%. In some embodiments, the concentration of sucrose is 25%. In some embodiments, the concentration of sucrose is 30%. In some embodiments, the concentration of sucrose is 35%. In some embodiments, the concentration of sucrose is 40%.

[0222] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding maltose as the lyoprotectant in step ii). In some embodiments, maltose isadded at a concentration of 0%-40%. In some embodiments, maltose is added at a concentration of 5%-20%. In some embodiments, maltose is added at a concentration of 5%- 10%. In some embodiments, maltose is added at a concentration of 10%-l 5%. In some embodiments, maltose is added at a concentration of 15%-20%. In some embodiments, maltose is added at a concentration of 20%-25%. In some embodiments, maltose is added at a concentration of 25%-30%. In some embodiments, maltose is added at a concentration of 30%-35%. In some embodiments, maltose is added at a concentration of 35%-40%. In some embodiments, the concentration of maltose is 5%. In some embodiments, the concentration of maltose is 10%. In some embodiments, the concentration of maltose is 15%. In some embodiments, the concentration of maltose is 20%. In some embodiments, the concentration of maltose is 25%. In some embodiments, the concentration of maltose is 30%. In some embodiments, the concentration of maltose is 35%. In some embodiments, the concentration of maltose is 40%.

[0223] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding trehalose or lactose or glucose as the lyoprotectant in step ii). In some embodiments, trehalose or lactose or glucose is added at a concentration of 0%-40%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 5%-20%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 5%-l 0%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 10%-l 5%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 15%-20%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 20%- 25%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 25%-30%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 30%-35%. In some embodiments, trehalose or lactose or glucose is added at a concentration of 35%-40%. In some embodiments, the concentration of trehalose or lactose or glucose is 5%. In some embodiments, the concentration of trehalose or lactose or glucose is 10%. In some embodiments, the concentration of trehalose or lactose or glucose is 15%. In some embodiments, the concentration of trehalose or lactose or glucose is 20%. In some embodiments, the concentration of trehalose or lactose or glucose is 25%. In some embodiments, the concentration of trehalose or lactose or glucose is 30%. In some embodiments, the concentration of trehalose or lactose or glucose is 35%. In some embodiments, the concentration of trehalose or lactose or glucose is 40%.

[0224] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyoprotectant comprises an amino acid. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is PBS. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is phosphate. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is a Tris buffer. In some embodiments, the lyoprotectant comprises an amino acid and the buffer is an acetate buffer. In some embodiments, the amino acid is lysine, arginine, leucine, glutamic acid, or any combination thereof. In some embodiments, the amino acid is lysine. In some embodiments, the amino acid is arginine. In some embodiments, the amino acid is leucine. In some embodiments, the amino acid is glutamic acid.

[0225] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyoprotectant comprises lysine. In some embodiments, lysine is added at a concentration of 0%-40%. In some embodiments, lysine is added at a concentration of 5%- 20%. In some embodiments, lysine is added at a concentration of 5%-10%. In some embodiments, lysine is added at a concentration of 10%-l 5%. In some embodiments, lysine is added at a concentration of 15%-20%. In some embodiments, lysine is added at a concentration of 20%-25%. In some embodiments, lysine is added at a concentration of 25%-30%. In some embodiments, lysine is added at a concentration of 30%-35%. In some embodiments, lysine is added at a concentration of 35%-40%. In some embodiments, the concentration of lysine is 5%. In some embodiments, the concentration of lysine is 10%. In some embodiments, the concentration of lysine is 15%. In some embodiments, the concentration of lysine is 20%. In some embodiments, the concentration of lysine is 25%. In some embodiments, the concentration of lysine is 30%. In some embodiments, the concentration of lysine is 35%. In some embodiments, the concentration of lysine is 40%.

[0226] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding an excipient comprising a lyoprotectant and a buffer in step ii). In some embodiments, the lyoprotectant comprises arginine or leucine or glutamic acid. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 0%-40%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 5%- 20%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 5%-l 0%. In some embodiments, arginine or leucine or glutamic acid is added at aconcentration of 10%- 15%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 15%-20%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 20%-25%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 25%-30%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 30%-35%. In some embodiments, arginine or leucine or glutamic acid is added at a concentration of 35%-40%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 5%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 10%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 15%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 20%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 25%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 30%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 35%. In some embodiments, the concentration of arginine or leucine or glutamic acid is 40%.

[0227] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding a buffer in step ii). As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. In some embodiments, the buffer of this invention has a pH from about 4.0 to about 8.0, for example from about 5.0 to about 7.0, e.g. from about 5.8 to about 6.2. Examples of buffers include, but are not limited to, acetate, succinate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers. In one embodiment herein, the buffer is a histidine buffer. In some embodiments, the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

[0228] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding PBS in step ii). In some embodiments, the PBS is 0.5x PBS. In some embodiments, the PBS is lx PBS. In some embodiments, the PBS is 2x PBS. In some embodiments, the PBS is 3x PBS. In some embodiments, the PBS is 4x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is lOx PBS. In some embodiments, the PBS is 20x PBS. In some embodiments, the PBS is 5x PBS. In some embodiments, the PBS is 50x PBS. In some embodiments, the PBS has a pH of about 5.8 to about 7.8. In some embodiments, the PBS has a pH of about 5.8. In some embodiments, the PBS has a pH of about 6.0. In some embodiments, the PBS has a pH of about 7.0. In some embodiments, the PBS has a pH of about 7.4. In some embodiments, the PBS has a pH of about 7.6. In some embodiments, the PBS has a pH of about 7.8. In some embodiments, thePBS comprises 10 mM phosphate buffer. In some embodiments, the PBS comprises 0.9% saline.

[0229] The method of making a lyophilized formulation comprising a NA-LNP particle comprises adding Tris in step ii. In some embodiments, the Tris is Tris -EDTA (TE). In some embodiments, the Tris is Tris-Acetate-EDTA (TAE). In some embodiments, the Tris is Tris- HC1. In some embodiments, the Tris is Tris-Bor...

Claims

CLAIMS1. A lyophilized formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a lyoprotectant and a buffer; wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG).

2. The lyophilized formulation of claim 1, wherein the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

3. The lyophilized formulation of claim 1, wherein the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

4. The lyophilized formulation of any one of claims 1-3, wherein the buffer is phosphate-buffered saline (PBS), a phosphate buffer, a Tris buffer or an acetate buffer.

5. The lyophilized formulation of claim 4, wherein the acetate buffer is a sodium acetate trihydrate buffer.

6. The lyophilized formulation of any one of claims 1-5, wherein the nucleic acid comprises a deoxyribonucleic acid (DNA).

7. The lyophilized formulation of claim 6, wherein the DNA is a circular DNA or a linear DNA.

8. The lyophilized formulation of claim 6, wherein the DNA is a plasmid DNA.

9. The lyophilized formulation of claim 6, wherein the DNA is a genomic DNA.

10. The lyophilized formulation of claim 6, wherein the DNA is a synthetic DNA.

11. The lyophilized formulation of claim 10, wherein the synthetic DNA is a cDNA.

12. The lyophilized formulation of any one of claims 1-5, wherein the nucleic acid comprises a ribonucleic acid (RNA).

13. The lyophilized formulation of claim 12, wherein the RNA is a circular RNA or a linear RNA.

14. The lyophilized formulation of claim 12, wherein the RNA is messenger RNA.

15. The lyophilized formulation of claim 12, wherein the RNA is a micro RNA (miRNA).

16. The lyophilized formulation of claim 12, wherein the RNA is a small inhibitory RNA (siRNA).

17. The lyophilized formulation of claim 12, wherein the RNA is a guide RNA (gRNA).

18. The lyophilized formulation of claim 6 or 12, wherein the DNA or RNA is an antisense oligonucleotide.

19. The lyophilized formulation of any one of claims 1-18, wherein the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof.

20. The lyophilized formulation of claim 19, wherein the ionizable lipid comprises SM- 102.

21. The lyophilized formulation of claim 19, wherein the phospholipid comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).

22. The lyophilized formulation of claim 19, wherein the PEGylated lipid comprises DMG-PEG2k.

23. The lyophilized formulation of any one of claims 1-22, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the lyoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v).

24. The lyophilized formulation of any one of claims 1-22, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the lyoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 20% or 30% weight per volume (w / v); optionally wherein the lyoprotectant is PVP and wherein the lyoprotectant is 5% PVP-K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25; optionally wherein the lyoprotectant is PEG and wherein the lyoprotectant is 10% PEG-1500.

25. The lyophilized formulation of any one of claims 1-24 wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or wherein the buffer is a phosphate buffer.

26. The lyophilized formulation of any one of claims 1-24 wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM.

27. The lyophilized formulation of any one of claims 1-24, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

28. The lyophilized formulation of any one of claims 1-24, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM.

29. The lyophilized formulation of any one of claims 1-24, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

30. The lyophilized formulation of any one of claims 1-29, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is between pH 4.0 and pH 8.0.

31. The lyophilized formulation of any one of claims 1-29, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 4.0.

32. The lyophilized formulation of any one of claims 1-29, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

33. The lyophilized formulation of any one of claims 1-32, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml.

34. The lyophilized formulation of any one of claims 1-32, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml or 50 pg / ml.

35. The lyophilized formulation of any one of claims 1-34, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm.

36. The lyophilized formulation of any one of claims 1-34, wherein the lyophilized formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA-LNP particle in the aqueous NA-LNP formulation is about 100 nm.

37. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PVP and the buffer is PBS or a phosphate buffer.

38. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PVP and the buffer is a Tris buffer.

39. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PVP and the buffer is an acetate buffer.

40. The lyophilized formulation of claim 37, 38, or 39, wherein the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

41. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PEG and the buffer is PBS or a phosphate buffer.

42. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PEG and the buffer is a Tris buffer.

43. The lyophilized formulation of claim 1, wherein the lyoprotectant is a PEG and the buffer is an acetate buffer.

44. The lyophilized formulation of claim 41, 42, or 43, wherein the PEG is PEG200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG- 12000 or PEG-20000.

45. A reconstituted formulation comprising a NA-LNP particle, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation of any one of claims 1-44 with a liquid.

46. The reconstituted formulation of claim 45, wherein the liquid is water or a buffer.

47. A method of making a lyophilized formulation comprising a NA-LNP particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, the method comprising: i. generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii. adding an excipient comprising a lyoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation; wherein the lyoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG); and iii. lyophilizing the aqueous NA-LNP formulation to produce the lyophilized formulation.

48. The method of claim 47, wherein the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

49. The method of claim 47 or 48, further comprising, after step (ii) and before step (iii): a) freezing the aqueous NA-LNP formulation to obtain a frozen formulation, and b) removing water from the formulation by sublimation, wherein the freezing of step a) occurs at -20 degrees Celsius or -80 degrees Celsius.

50. The method of any one of claims 47-49, wherein the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof.

51. The method of claim 50, wherein the ionizable lipid comprises SM-102.

52. The method of claim 50, wherein the phospholipid comprises 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC).

53. The method of claim 50, wherein the PEGylated lipid comprises DMG-PEG2k.

54. The method of any one of claims 47-53, wherein the total concentration of the one or more lipids in the organic phase is between 0.1 mM to 20 mM.

55. The method of any one of claims 47-53, wherein the concentration of each of the one or more lipids in the organic phase is 0.1 mM, 0.125 mM, 0.2 mM, 0.5 mM, 1 mM, 6.25 mM, 10 mM, 12.5 mM, or 20 mM.

56. The method of any one of claims 47-55, wherein PVP is PVP-K12, PVP-K15, PVP- K30, PVP-K60, PVP-K90, or PVP-K120.

57. The method of any one of claims 47-55, wherein the PEG is PEG-200, PEG-400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

58. The method of any one of claims 47-57, wherein the nucleic acid comprises a deoxyribonucleic acid (DNA).

59. The method of claim 58, wherein the DNA is a circular DNA or a linear DNA.

60. The method of claim 58, wherein the DNA is a plasmid DNA.

61. The method of claim 58, wherein the DNA is a genomic DNA.

62. The method of claim 58, wherein the DNA is a synthetic DNA.

63. The method of claim 62, wherein the synthetic DNA is a cDNA.

64. The method of any one of claims 47-57, wherein the nucleic acid comprises a ribonucleic acid (RNA).

65. The method of claim 64, wherein the RNA is messenger RNA.

66. The method of claim 58 or 64, wherein the DNA or RNA is an antisense oligonucleotide.

67. The method of any one of claims 47-66, wherein the concentration of the lyoprotectant in step (ii) is between 5% and 40% weight per volume (w / v).

68. The method of any one of claims 47-66, wherein the concentration of the lyoprotectant in step (ii) is 5%, 10%, 20% or 30% weight per volume (w / v); optionally wherein the lyoprotectant is PVP and wherein the lyoprotectant is 5% PVP- K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25; optionally wherein the lyoprotectant is PEG and wherein the lyoprotectant is 10% PEG- 1500.

69. The method of any one of claims 47-68 wherein the buffer is an acetate buffer, and wherein the concentration of the acetate buffer in step (ii) is 10 mM, 20 mM or 50 mM.

70. The method of claim 69, wherein the acetate buffer is a sodium acetate trihydrate buffer.

71. The method of any one of claims 47-68, wherein the buffer is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or wherein the buffer is a phosphate buffer.

72. The method of any one of claims 47-68, wherein the buffer is a Tris buffer, and wherein the concentration of the Tris buffer is 10 mM, 20 mM or 50 mM.

73. The method of any one of claims 47-72, wherein the pH of the buffer in step (ii) is between pH 4.0 and pH 8.0.

74. The method of any one of claims 47-72, wherein the pH of the buffer is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

75. The method of any one of claims 47-74, wherein the organic phase and the aqueous phase are mixed at a flow rate ratio of 1 :3 of organic:aqueous phase.

76. The method of any one of claims 47-75, wherein the method further comprises reconstituting the lyophilized formulation with a liquid to produce a reconstituted formulation comprising the NA-LNP particle.

77. The method of claim 76, wherein the diameter of the NA-LNP particle in the reconstituted formulation is between 50 nm and 300 nm.

78. The method of claim 76, wherein the diameter of the NA-LNP particle in the reconstituted formulation is about 100 nm.

79. The method of claim 76, wherein the diameter of the NA-LNP particle in the reconstituted formulation is no more than 10% larger, 20% larger or 30% larger than the diameter of the NA-LNP particle in the NA-LNP formulation in step ii).

80. The method of claim 76, wherein the percent encapsulation efficiency (%EE) of the nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower, 20% lower, or 30% lower than the percent encapsulation efficiency (%EE) of the of the nucleic acid in the NA-LNP particle in the NA-LNP formulation in step ii).

81. The method of claim 80, wherein the percent encapsulation efficiency (%EE) is calculated asSoluble nucleic acid concentration%EE = (1 - — - - - — - - -:- ) x 100%Total nucleic acid concentration82. The method of claim 76, wherein the amount of intact nucleic acid in the NA-LNP particle in the reconstituted formulation is no more than 10% lower, 20% lower, or 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the NA- LNP formulation in step ii).

83. An in vitro or ex vivo method for introducing a nucleic acid encapsulated in a lipid nanoparticle into a cell, the method comprising contacting the cell with a reconstituted formulation, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation of any one of claims 1-44 with a liquid.

84. The method of claim 83, wherein the liquid is water or a buffer.

85. The method of claim 83 or 84, wherein the nucleic acid is expressed in the cells.

86. The method of claim 83, 84, or 85, wherein the cells do not produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.

87. A frozen formulation comprising a nucleic acid (NA)-lipid nanoparticle (LNP) particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, wherein the formulation further comprises an excipient comprising a cryoprotectant and a buffer; wherein the cryoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG).

88. The frozen formulation of claim 87, wherein the PVP is PVP-K12, PVP -KI 5, PVP- K30, PVP-K60, PVP-K90, or PVP-K120.

89. The frozen formulation of claim 88, wherein the PEG is PEG-200, PEG-400, PEG- 600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG- 20000.

90. The frozen formulation of any one of claims 87-89, wherein the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

91. The frozen formulation of claim 90, wherein the acetate buffer is a sodium acetate trihydrate buffer.

92. The frozen formulation of any one of claims 87-91, wherein the nucleic acid comprises a deoxyribonucleic acid (DNA).

93. The frozen formulation of claim 92, wherein the DNA is a circular DNA or a linear DNA.

94. The frozen formulation of claim 92, wherein the DNA is a plasmid DNA.

95. The frozen formulation of claim 92, wherein the DNA is a genomic DNA.

96. The frozen formulation of claim 92, wherein the DNA is a synthetic DNA.

97. The frozen formulation of claim 96, wherein the synthetic DNA is a cDNA.

98. The frozen formulation of any one of claims 87-91, wherein the nucleic acid comprises a ribonucleic acid (RNA).

99. The frozen formulation of claim 98, wherein the RNA is a circular RNA or a linear RNA.

100. The frozen formulation of claim 98, wherein the RNA is messenger RNA.

101. The frozen formulation of claim 98, wherein the RNA is a micro RNA (miRNA).

102. The frozen formulation of claim 98, wherein the RNA is a small inhibitory RNA (siRNA).

103. The frozen formulation of claim 98, wherein the RNA is a guide RNA (gRNA).

104. The frozen formulation of claim 92 or 98, wherein the DNA or RNA is an antisense oligonucleotide.

105. The frozen formulation of any one of claims 87-104, wherein the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof.

106. The frozen formulation of claim 105, wherein the ionizable lipid comprises SM-102.

107. The frozen formulation of claim 105, wherein the phospholipid comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).

108. The frozen formulation of claim 105, wherein the PEGylated lipid comprises DMG- PEG2k.

109. The frozen formulation of any one of claims 87-108, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the cryoprotectant in the aqueous NA-LNP formulation is between 5% and 40% weight per volume (w / v).

110. The frozen formulation of any one of claims 87-108, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the concentration of the cryoprotectant in the aqueous NA-LNP formulation is 5%, 10%, 20% or 30% weight per volume (w / v); optionally wherein the cryoprotectant is PVP and wherein the lyoprotectant is 5% PVP-K12, 10% PVP-K12, 20% PVP-K15, 5% PVP-K17, 10% PVP-K17, 20% PVP=K17, 5% PVP-K25, 10% PVP-K25, or 20% PVP-K25; optionally wherein the cryoprotectant is PEG and wherein the lyoprotectant is 10% PEG-1500.

111. The frozen formulation of any one of claims 87-110, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or wherein the buffer is a phosphate buffer.

112. The frozen formulation of any one of claims 87-110, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM.

113. The frozen formulation of any one of claims 87-110, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is a Tris buffer, and wherein the concentration of the Tris buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

114. The frozen formulation of any one of claims 87-110, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueousNA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is between 5 mM and 100 mM.

115. The frozen formulation of any one of claims 87-110, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the buffer in the aqueous NA-LNP formulation is an acetate buffer, and wherein the concentration of the acetate buffer in the aqueous NA-LNP formulation is 10 mM, 20 mM or 50 mM.

116. The frozen formulation of any one of claims 87-115, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is between pH 4.0 and pH 8.0.

117. The frozen formulation of any one of claims 87-115, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, and wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 4.0.

118. The frozen formulation of any one of claims 87-115, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the pH of the buffer in the aqueous NA-LNP formulation is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

119. The frozen formulation of any one of claims 87-118, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is between 5 pg / ml and 100 pg / ml.

120. The frozen formulation of any one of claims 87-118, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the concentration of the nucleic acid in the aqueous NA-LNP formulation is 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml or 50 pg / ml.

121. The frozen formulation of any one of claims 87-120, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is between 50 nm and 300 nm.

122. The frozen formulation of any one of claims 87-120, wherein the frozen formulation is obtained from an aqueous NA-LNP formulation, wherein the diameter of the NA- LNP particle in the aqueous NA-LNP formulation is about 100 nm.

123. The frozen formulation of claim 87, wherein the cryoprotectant is a PVP and the buffer is PBS or a phosphate buffer.

124. The frozen formulation of claim 87, wherein the cryoprotectant is a PVP and the buffer is a Tris buffer.

125. The frozen formulation of claim 87, wherein the cryoprotectant is a PVP and the buffer is an acetate buffer.

126. The frozen formulation of claim 123, 124, or 125, wherein the PVP is PVP-K12, PVP-K15, PVP-K17, PVP-K25, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

127. The frozen formulation of claim 87, wherein the cryoprotectant is a PEG and the buffer is PBS or a phosphate buffer.

128. The frozen formulation of claim 87, wherein the cryoprotectant is a PEG and the buffer is a Tris buffer.

129. The frozen formulation of claim 87, wherein the cryoprotectant is a PEG and the buffer is an acetate buffer.

130. The frozen formulation of claim 127, 128, or 129, wherein the PEG is PEG200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

131. The frozen formulation of claims 87-130, wherein the frozen formulation is frozen at - 20 degrees Celsius or -80 degrees Celsius.

132. A thawed formulation comprising a NA-LNP particle, wherein the thawed formulation is obtained by thawing the frozen formulation of any one of claims 87-133. The thawed formulation of claim 132, wherein the thawed formulation is obtained by at least one, at least two, or at least three cycles comprising (i) freezing the thawed NA-LNP formulation to obtain a frozen NA-LNP formulation, followed by (ii) thawing the frozen formulation of (i).

134. The thawed formulation of claim 133, wherein the freezing of (i) occurs at -20 degrees Celsius or -80 degrees Celsius to obtain a frozen formulation.

135. The thawed formulation of claim 133 or 134, wherein the frozen formulation is thawed at about 20 degrees Celsius to 25 degrees Celsius.

136. A method of making a frozen formulation comprising a NA-LNP particle, wherein the NA-LNP particle comprises a nucleic acid encapsulated in the lipid nanoparticle, the method comprising: i. generating a NA-LNP particle by mixing an organic phase with an aqueous phase, wherein the organic phase comprises one or more lipids and the aqueous phase comprises a nucleic acid; ii. adding an excipient comprising a cryoprotectant and a buffer to the NA-LNP particle to produce an aqueous NA-LNP formulation; wherein the cryoprotectant is a polyvinylpyrrolidone (PVP) or a polyethylene glycol (PEG); and iii. freezing the aqueous NA-LNP formulation to obtain a frozen formulation.

137. The method of claim 136, wherein the buffer is PBS, a phosphate buffer, a Tris buffer, or an acetate buffer.

138. The method of claim 136 or 137, wherein the freezing of step (iii) occurs at -20 degrees Celsius or -80 degrees Celsius.

139. The method of claim 137 or 138, further comprising: step (iv) thawing the frozen formulation to obtain a thawed formulation, then freezing the thawed formulation.

140. The method of claim 139, wherein step (iv) is repeated at least one, at least two, or at least three times.

141. The method of claim 139 or 140, wherein the thawing of step (iv) occurs at about 20 to 25 degrees Celsius.

142. The method of claim any one of claims 136-141, wherein the LNP comprises a cholesterol, an ionizable lipid, a phospholipid, a PEGylated lipid, or any combination thereof.

143. The method of claim 142, wherein the ionizable lipid comprises SM-102.

144. The method of claim 142, wherein the phospholipid comprises 1,2-distearoyl-sn- glycero-3 -phosphocholine (DSPC).

145. The method of claim 142, wherein the PEGylated lipid comprises DMG-PEG2k.

146. The method of claim any one of claims 136-145, wherein the total concentration of the one or more lipids in the organic phase is between 0.1 mM to 20 mM.

147. The method of claim any one of claims 136-145, wherein the concentration of each of the one or more lipids in the organic phase is 0.1 mM, 0.125 mM, 0.2 mM, 0.5 mM, 1 mM, 6.25 mM, 10 mM, 12.5 mM, or 20 mM.

148. The method of claim any one of claims 136-147, wherein the PVP is PVP-K12, PVP- K15, PVP-K30, PVP-K60, PVP-K90, or PVP-K120.

149. The method of claim any one of claims 136-147, wherein the PEG is PEG-200, PEG- 400, PEG-600, PEG-1500, PEG-3350, PEG-4000, PEG-6000, PEG-8000, PEG-12000 or PEG-20000.

150. The method of claim any one of claims 136-149, wherein the nucleic acid comprises a deoxyribonucleic acid (DNA).

151. The method of claim 150, wherein the DNA is a circular DNA or a linear DNA.

152. The method of claim 150, wherein the DNA is a plasmid DNA.

153. The method of claim 150, wherein the DNA is a genomic DNA.

154. The method of claim 150, wherein the DNA is a synthetic DNA.

155. The method of claim 154, wherein the synthetic DNA is a cDNA.

156. The method of claim any one of claims 136-149, wherein the nucleic acid comprises a ribonucleic acid (RNA).

157. The method of claim 156, wherein the RNA is messenger RNA.

158. The method of claim 150 or 156, wherein the DNA or RNA is an antisense oligonucleotide.

159. The method of any one of claims 136-158, wherein the concentration of the cryoprotectant in step (ii) is between 5% and 40% weight per volume (w / v).

160. The method of any one of claims 136-158, wherein the concentration of the cryoprotectant in step (ii) is 5%, 10%, 20% or 30% weight per volume (w / v).

161. The method of any one of claims 136-160, wherein the buffer is an acetate buffer, and wherein the concentration of the acetate buffer in step (ii) is 10 mM, 20 mM or 50 mM.

162. The method of claim 161, wherein the acetate buffer is a sodium acetate trihydrate buffer.

163. The method of any one of claims 136-160, wherein the buffer is PBS, and the PBS is lx PBS, 5x PBS or lOx PBS, or wherein the buffer is a phosphate buffer.

164. The method of any one of claims 136-160, wherein the buffer is a Tris buffer, and wherein the concentration of the Tris buffer is 10 mM, 20 mM or 50 mM.

165. The method of any one of claims 136-164, wherein the pH of the buffer in step (ii) is between pH 4.0 and pH 8.0.

166. The method of any one of claims 136-164, wherein the pH of the buffer is pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7 or pH 7.8.

167. The method of any one of claims 136-166, wherein the organic phase and the aqueous phase are mixed at a flow rate ratio of 3 : 1.

168. The method of any one of claims 136-167, wherein the method further comprises thawing the frozen formulation to produce a thawed formulation comprising the NA- LNP particle.

169. The method of claim 168, wherein the diameter of the NA-LNP particle in the thawed formulation is between 50 nm and 300 nm.

170. The method of claim 168, wherein the diameter of the NA-LNP particle in the thawed formulation is about 100 nm.

171. The method of claim 168, wherein the diameter of the NA-LNP particle in the thawed formulation is no more than 10% larger, 20% larger or 30% larger than the diameter of the NA-LNP particle in the NA-LNP formulation in step ii).

172. The method of claim 168, wherein the percent encapsulation efficiency (%EE) of the nucleic acid in the NA-LNP particle in the thawed formulation is no more than 10% lower, 20% lower, or 30% lower than the percent encapsulation efficiency (%EE) of the of the nucleic acid in the NA-LNP particle in the NA-LNP formulation in step ii).

173. The method of claim 172, wherein the percent encapsulation efficiency (%EE) is calculated asSoluble nucleic acid concentration%EE = (1 - — - - - — - - -;- ) x 100%Total nucleic acid concentration174. The method of claim 168, wherein the amount of intact nucleic acid in the NA-LNP particle in the thawed formulation is no more than 10% lower, 20% lower, or 30% lower than the amount of intact nucleic acid in the NA-LNP particle in the NA-LNP formulation in step ii).

175. An in vitro or ex vivo method for introducing a nucleic acid encapsulated in a lipid nanoparticle into a cell, the method comprising contacting the cell with a thawed formulation, wherein the thawed formulation is obtained by thawing the frozen formulation of any one of claims 87-131.

176. The method of claim 175, wherein the frozen formulation is thawed at 20 degrees Celsius to 25 degrees Celsius.

177. The method of claim 175 or 176, wherein the nucleic acid is expressed in the cells.

178. The method of claim 175, 176, or 177, wherein the cells do not produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.

179. Use of a reconstituted formulation comprising a nucleic acid encapsulated in a lipid nanoparticle in the manufacture of a medicament for use in a method of introducing the nucleic acid encapsulated in a lipid nanoparticle into a cell, wherein the method comprises contacting the cell with the reconstituted formulation, wherein the reconstituted formulation is obtained by reconstituting the lyophilized formulation of any one of claims 1-44 with a liquid.

180. The method of claim 179, wherein the liquid is water or a buffer.

181. The method of claim 179 or 180, wherein the nucleic acid is expressed in the cells.

182. The method of claim 179, 180, or 181, wherein the cells do not produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.

183. Use of a thawed formulation comprising a nucleic acid encapsulated in a lipid nanoparticle in the manufacture of a medicament for use in a method of introducing the nucleic acid encapsulated in a lipid nanoparticle into a cell, wherein the method comprises contacting the cell with the thawed formulation, wherein the thawed formulation is obtained by thawing the frozen formulation of any one of claims 87- 131.

184. The method of claim 183, wherein the frozen formulation is thawed at 20 degrees Celsius to 25 degrees Celsius.

185. The method of claim 183 or 184, wherein the nucleic acid is expressed in the cells.

186. The method of claim 183, 184, or 185, wherein the cells do not produce an increased amount of cytokines or chemokines compared to a cell that is contacted with the aqueous NA-LNP formulation.

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