Lipid nanoparticles, methods of making lipid nanoparticles, and uses thereof

Abstract

The present disclosure provides, among other things, improved lipid nanoparticles and improved methods of making lipid nanoparticles.

Description

[0001] PATENT

[0002] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0003] LIPID NANOPARTICLES, METHODS OF MAKING LIPID NANOPARTICLES, AND USES THEREOF

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] [1] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 753,807, filed February 4, 2025, which is hereby incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] [2] Lipid nanoparticles are used to deliver a variety of bioactive molecules including nucleic acids to target cells and tissues. Lipid nanoparticle delivery provides several advantages such as increased drug stability, bioavailability, and distribution to target tissues over conventional methods. For example, nucleic acid cargo encapsulated within LNPs are protected from enzymatic degradation during the delivery process and are efficiently delivered to cells, where the therapeutic cargo is released.

[0008] [3] There remains a need for efficient and cost-effective methods of making stable lipid nanoparticles with improved size, polydispersity and efficacy that can accommodate large nucleic acid cargo for therapeutic applications, for example, gene editing.

[0009] SUMMARY OF THE INVENTION

[0010] [4] The present disclosure provides, among other things, lipid nanoparticles, methods of making lipid nanoparticles, and lipid nanoparticles made by the methods of the present invention.

[0011] [5] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0012] (a) a nucleic acid cargo;

[0013] (b) an ionizable lipid comprising from 51 mol % to 60 mol % of the total lipid present in the particle;

[0014] (c) a phospholipid comprising from 7 mol % to 10 mol % of the total lipid present in the particle;PATENT

[0015] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0016] (d) a cholesterol lipid, or derivative thereof, comprising from 30 mol % to 40 mol % of the total lipid present in the particle; and

[0017] (e) a PEG lipid comprising from 1 mol % to 5 mol % of the total lipid present in the particle.

[0018] [6] In some embodiments, the ionizable lipid comprises from about 54 mol % to about 57 mol % of the total lipid present in the LNP.

[0019] [7] In some embodiments, the phospholipid comprises from about 7 mol% to about 8 mol% of the total lipid present in the LNP.

[0020] [8] In some embodiments, the cholesterol lipid or derivative thereof comprises from about 35 mol % to about 40 mol % of the total lipid present in the LNP.

[0021] [9] In some embodiments, the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0022]

[0010] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0023]

[0011] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

[0024]

[0012] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0025] (a) a nucleic acid cargo;

[0026] (b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;

[0027] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0028] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; andPATENT

[0029] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0030] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP,

[0031] wherein the LNP is prepared by mixing the nucleic acid cargo, the ionizable lipid, the phospholipid, the cholesterol lipid or derivative thereof, and the PEG lipid at a pH of between 5.9 and 6.2.

[0032]

[0013] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the particle.

[0033]

[0014] In some embodiments, the phospholipid comprises from about 9.5 mol% to about 10 mol% of the total lipid present in the particle.

[0034]

[0015] In some embodiments, the cholesterol lipid or derivative thereof comprises from about 35 mol % to about 40 mol % of the total lipid present in the particle.

[0035]

[0016] In some embodiments, the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the particle.

[0036]

[0017] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0037]

[0018] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0038]

[0019] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0039] (a) a nucleic acid cargo;

[0040] (b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;

[0041] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0042] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % toPATENT

[0043] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0044] about 40 mol % of the total lipid present in the LNP; and

[0045] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle

[0046] wherein the hydrodynamic diameter of the LNP increases by less than about 10 nm after at least one freeze thaw cycle.

[0047]

[0020] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 10 nm after two freeze thaw cycles.

[0048]

[0021] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 10 nm after three freeze thaw cycles.

[0049]

[0022] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0050]

[0023] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

[0051]

[0024] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0052] (a) a nucleic acid cargo;

[0053] (b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;

[0054] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0055] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and

[0056] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particlePATENT

[0057] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0058] wherein the hydrodynamic diameter of the LNP increases by less than about 20% after at least one freeze thaw cycle.

[0059]

[0025] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 20% after two freeze thaw cycles.

[0060]

[0026] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 20% after three freeze thaw cycles.

[0061]

[0027] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15% after three freeze thaw cycles.

[0062]

[0028] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 10% after three freeze thaw cycles.

[0063]

[0029] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 5% after three freeze thaw cycles.

[0064]

[0030] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the particle; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0065]

[0031] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the particle; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

[0066]

[0032] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0067] (a) a nucleic acid cargo;

[0068] (b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;

[0069] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0070] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; andPATENT

[0071] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0072] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle

[0073] wherein the hydrodynamic diameter of the LNP increases by less than about 20% after storage at -20°C for up to 4 weeks, or

[0074] wherein the hydrodynamic diameter of the LNP increases by less than about 10% after storage at -80°C for up to 3 months.

[0075]

[0033] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15%, about 10%, about 5%, or about 1% after storage at -20°C for up to 4 weeks.

[0076]

[0034] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after storage at -80°C for up to 3 months.

[0077]

[0035] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0078]

[0036] In some embodiments, the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

[0079]

[0037] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0080] (a) a nucleic acid cargo;

[0081] (b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;

[0082] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0083] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; andPATENT

[0084] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0085] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle

[0086] wherein the hydrodynamic diameter of the LNP increases by less than about 15 nm after at least one freeze thaw cycle.

[0087]

[0038] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15 nm after two freeze thaw cycles.

[0088]

[0039] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15 nm after three freeze thaw cycles.

[0089]

[0040] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0090]

[0041] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0091] (a) a nucleic acid cargo;

[0092] (b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;

[0093] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0094] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and

[0095] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle

[0096] wherein the hydrodynamic diameter of the LNP increases by less than about 20% after at least one freeze thaw cycle.

[0097]

[0042] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 20% after two freeze thaw cycles.

[0098]

[0043] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 20% after three freeze thaw cycles.PATENT

[0099] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0100]

[0044] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15% after three freeze thaw cycles.

[0101]

[0045] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 10% after three freeze thaw cycles.

[0102]

[0046] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 5% after three freeze thaw cycles.

[0103]

[0047] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0104]

[0048] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0105] (a) a nucleic acid cargo;

[0106] (b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;

[0107] (c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;

[0108] (d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and

[0109] (e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle

[0110] wherein the hydrodynamic diameter of the LNP increases by less than about 20% after storage at -20°C for up to 4 weeks, or

[0111] wherein the hydrodynamic diameter of the LNP increases by less than about 10% after storage at -80°C for up to 3 months.

[0112]

[0049] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 15%, about 10%, about 5%, or about 1% after storage at -20°C for up to 4 weeks.PATENT

[0113] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0114]

[0050] In some embodiments, the hydrodynamic diameter of the LNP increases by less than about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at -80°C for up to 3 months.

[0115]

[0051] In some embodiments, the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

[0116]

[0052] In some embodiments, the encapsulation efficiency after storage is greater than about 90%.

[0117]

[0053] In some embodiments, the encapsulation efficiency after storage is greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0118]

[0054] In some embodiments, the ionizable lipid is selected from Table 1.

[0119]

[0055] In some embodiments, the ionizable lipid is selected from Table 2.

[0120]

[0056] In some embodiments, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

[0121]

[0057] In some embodiments, the phospholipid comprises distearoylphosphatidylcholine (DSPC).

[0122]

[0058] In some embodiments, the PEG lipid comprises PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE, or any combination thereof.

[0123]

[0059] In some embodiments, the PEG lipid comprises a PEG-dimyristylglycerol (PEG-DMG) conjugate.

[0124]

[0060] In some embodiments, the nucleic acid cargo comprises between about 3000 to about 8000 nucleotides (nts).PATENT

[0125] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0126]

[0061] In some embodiments, the nucleic acid cargo comprises between about 5000 to about 7000 nucleotides.

[0127]

[0062] In some embodiments, the nucleic acid cargo comprises a guide RNA and an mRNA encoding a CRISPR base editor.

[0128]

[0063] In some embodiments, the N:P ratio of between about 10:1 to about 5:1.

[0129]

[0064] In some embodiments, the LNP has an average hydrodynamic diameter between about 40 nm to about 100 nm.

[0130]

[0065] In some embodiments, the LNP has an average hydrodynamic diameter between about 50 nm to about 70 nm.

[0131]

[0066] In some embodiments, the polydispersity index (PDI) is less than about 0.1.

[0132]

[0067] In some embodiments, the polydispersity index (PDI) is less than about 0.05.

[0133]

[0068] In some aspects, the hydrodynamic diameter of the LNP increases by less than about 10 nm after at least one to three freeze thaw cycles. In some aspects, the hydrodynamic diameter of the LNP increases by less than about 5 to 20% after at least one to three freeze thaw cycles. In some aspect, the hydrodynamic diameter of the LNP increases by less than about 5 to 20% after storage at -20°C for up to 4 weeks or after storage at -80°C for up to 3 months.

[0134]

[0069] In one aspect, the present disclosure features a lipid nanoparticle (LNP) comprising:

[0135] (a) a nucleic acid cargo;

[0136] (b) an ionizable lipid comprising from 47 mol % to 60 mol % of the total lipid present in the particle;

[0137] (c) a phospholipid comprising from 7 mol % to 10 mol % of the total lipid present in the particle;

[0138] (d) a cholesterol lipid, or derivative thereof, comprising from 30 mol % to 40 mol % of the total lipid present in the particle; and

[0139] (e) a PEG lipid comprising from 2 mol % to 3 mol % of the total lipid present in the particle.

[0140]

[0070] Among other things, the present disclosure recognizes that lipid nanoparticle (LNP) compositions undergo increase in the hydrodynamic diameter of the LNP. In some aspects, the hydrodynamic diameter of the LNP increases by less than about 10 nm after atPATENT

[0141] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0142] least one to three freeze thaw cycle. In some aspects, the hydrodynamic diameter of the LNP increases by less than about 5 to 20% after at least one to three freeze thaw cycle. In some aspects, the hydrodynamic diameter of the LNP increases by less than about 5 to 20% after storage at -20°C for up to 4 weeks or after storage at -80°C for up to 3 months. In some aspects, the encapsulation efficiency after storage is greater than 90%. In some aspect, the encapsulation efficiency after storage is greater than 91 to 99%.

[0143]

[0071] In one aspect, the present disclosure features a method of preparing a lipid nanoparticle (LNP) comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, wherein:

[0144] the first solution comprises one or more nucleic acids, and

[0145] the second solution comprises an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid;

[0146] wherein the mixing step is carried out to yield a mixed solution at a pH of between about 5.9 to 6.2,

[0147] thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0148]

[0072] In one aspect, the present disclosure features a method of preparing a lipid nanoparticle (LNP) comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, wherein:

[0149] the first solution comprises one or more nucleic acids,

[0150] the second solution comprises an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid;

[0151] wherein the mixing step is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid,

[0152] thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0153]

[0073] In some embodiments, the pH of the first solution is between pH 5.5 to pH 6.5 prior to the mixing step.

[0154]

[0074] In some embodiments, the method further comprises a step of adjusting the pH of the first solution and / or the second solution to within 0.5 units of each other prior to the mixing step.PATENT

[0155] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0156]

[0075] In some embodiments, the LNP comprises an N: P ratio of between about 10:1 to about 5:1.

[0157]

[0076] In some embodiments, the LNP has a hydrodynamic diameter between of about 40 nm to about 100 nm.

[0158]

[0077] In some embodiments, the LNP has a hydrodynamic diameter of about 50 nm to 70 nm.

[0159]

[0078] In some embodiments, the encapsulation efficiency of the LNP is at least about 90%.

[0160]

[0079] In some embodiments, the polydispersity index (PDI) of the LNP is less than about 0.1.

[0161]

[0080] In some embodiments, the first solution is an aqueous solution of water: ethanol in about 3: 1 (v / v) ratio, about 4: 1 (v / v) ratio, or about 2: 1 (v / v) ratio.

[0162]

[0081] In some embodiments, the second solution comprises:

[0163] (a) about 51 mol % to about 55 mol % of an ionizable lipid;

[0164] (b) about 7 mol % to about 10 mol % of a phospholipid;

[0165] (c) about 30 mol % to about 40 mol % of a cholesterol lipid, or derivative thereof; and (d) about 2 mol % to about 3 mol % of a PEG lipid.

[0166]

[0082] In some embodiments, the second solution comprises about 55 mol % of the ionizable lipid; about 7 mol % to about 8 mol% of the phospholipid; about 35% of the cholesterol lipid, or derivative thereof; and about 2 mol % to about 3 mol % the PEG lipid.

[0167]

[0083] In some embodiments, the second solution comprises about 55 mol % of the ionizable lipid; about 7.5 mol % to about 8 mol% of the phospholipid; about 35% of the cholesterol lipid, or derivative thereof; and about 2.5 mol % to about 3 mol % the PEG lipid.

[0168]

[0084] In some embodiments, the second solution comprises:

[0169] (a) about 47 mol % to about 60 mol % of an ionizable lipid;

[0170] (b) about 7 mol % to about 10 mol % of a phospholipid;

[0171] (c) about 30 mol % to about 40 mol % of a cholesterol lipid, or derivative thereof; and (d) about 2 mol % to about 3 mol % of a PEG lipid.PATENT

[0172] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0173]

[0085] In some embodiments, the second solution comprises about 47.5 mol % of the ionizable lipid; about 9.5 mol % to about 10 mol% of the phospholipid; about 40% of the cholesterol lipid, or derivative thereof; and about 2.5 mol % to about 3 mol % the PEG lipid.

[0174]

[0086] Among other things, the present disclosure recognizes that the method of preparing a lipid nanoparticle (LNP) composition comprises a mixing step that is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid to provide a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0175]

[0087] Other features, objects, and advantages of the present disclosure are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present disclosure, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the disclosure will become apparent to those skilled in the art from the detailed description.

[0176] BRIEF DESCRIPTION OF THE DRAWINGS

[0177]

[0088] The drawings are for illustration purposes and are in no way limiting.

[0178]

[0089] FIG. 1 shows a schematic of standard mixing setup for LNP formation, wherein the setup comprises continuous flow of RNA Packet (mRNA or gRNA, 50 mM citrate, and in an acidic pH environment) and Lipid Packet (ionizable lipid, DSPC, cholesterol, and PEG-lipid in ethanol), wherein both pools join at T-mixer and generate freshly formed LNP solution. The freshly formed LNP solution undergoes buffer exchange, ultra-centrifugation filtration, concentration of the LNP solution, and sterile filtration. The IDs of connecting tubing ranged from 0.01” to 0.04”. IL: ionizable lipid; Choi: Cholesterol.

[0179]

[0090] FIG. 2A-FIG. 2B shows cryoelectron micrographs showing morphologies of IL7 when the LNP is prepared with pH 6 of aqueous RNA mixture and with pH 4 of aqueous RNA mixture. FIG. 2A shows a cryoelectron micrograph showing morphology of TA7 (47.5 / 10 / 40 / 2.5; % Molar Ratio of IL7 / Cholesterol / DSPC / DMG-PEG2000 at pH 6) of IL7 compositional variant LNPs. FIG. FIG. 2B shows a cryoelectron micrograph showing morphology of TA17 (47.5 / 10 / 40 / 2.5; % Molar Ratio of IL7 / Cholesterol / DSPC / DMG-PEG2000 at pH 4) of IL 7 compositional variant LNPs, which is larger, less homogenous and shows more blebbing relative to TA7.

[0180]

[0091] FIG. 3A-FIG. 3C shows graphs of LNP size, encapsulation efficiency and stability (i.e., change in LNP size after three rounds of freeze and thaw) relative to mixing pHPATENT

[0181] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0182] ranging from pH 4 to 6.4 for exemplary LNPs comprising IL76 ionizable lipid. Circles represent conditions wherein the LNPs comprise 2.5% PEG. Triangles represent conditions wherein the LNPs comprise 2.1% PEG. Open circles and triangles represent conditions with flow rates of 15 mL / min. Closed circles and triangles represent conditions with flow rates of 10 mL / min. FIG. 3A is a graph comparing size of LNPs relative to mixing pH. FIG. 3B is a graph comparing encapsulation efficiency of LNPs relative to mixing pH. FIG. 3C is a graph comparing stability as measured by change in size of LNP particles after three cycles of freeze and thaw.

[0183]

[0092] FIG. 4A-FIG. 4D show heat maps of compositions comprising exemplary ionizable lipid and PEG lipid concentration ranges when mixed at pH 6 relative to control mixing at pH 4. FIG. 4A is a heat map comparing size of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG lipid and between 47.5-55% IL76 at mixing pH 6 relative to a control mixing pH 4. FIG. 4B is a heat map comparing encapsulation efficiency of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG lipid and between 47.5-55% IL76 at mixing pH 6 relative to a control mixing pH 4. FIG. 4C is a heat map comparing size growth of fresh LNPs subjected to three rounds of freeze-thaw of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG and between 47.5-55% IL76 lipid at mixing pH 6 relative to a control mixing pH 4. FIG. 4D is a heat map comparing size growth after storage for two weeks at -20C, and then subjected to three rounds of freeze-thaw of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG lipid and between 47.5-55% IL76 at mixing pH 6 relative to a control mixing pH 4.

[0184]

[0093] FIG. 5A-FIG. 5B shows cryoelectron micrographs showing morphology of prototype and stabilized IL76 LNPs. FIG. 5A shows a cryoelectron micrograph showing morphology of prototype IL76 LNPs. FIG. 5B shows a cryoelectron micrograph showing morphology of stabilized IL76 LNPs, which is smaller, more homogenous and shows less blebbing relative to prototype IL76 LNPs.

[0185]

[0094] FIG. 6A-FIG. 6B shows heat maps of compositions comprising exemplary ionizable lipid and PEG lipid concentration ranges when mixed at pH 6 relative to control mixing at pH 4. FIG. 6A is a heat map comparing size growth of fresh LNPs subjected to three rounds of freeze-thaw of IL477 LNPs comprising between 1.9-2.5% exemplary DMG-PEG lipid at mixing pH 6 relative to a control mixing pH 4. FIG. 6B is a heat map comparing size growth after storage for two weeks at -20 °C, and then subjected to three rounds ofPATENT

[0186] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0187] freeze-thaw of IL477 LNPs comprising between 1.9-2.5% exemplary DMG-PEG lipid at mixing pH 6 relative to a control mixing pH 4.

[0188]

[0095] FIG. 7A-FIG. 7B shows cryoelectron micrographs showing morphology of prototype and stabilized IL477 LNPs. FIG. 7A shows a cryoelectron micrograph showing morphology of prototype IL477 LNPs. FIG. 7B shows a cryoelectron micrograph showing morphology of stabilized IL477 LNPs, which is smaller, more homogenous and shows less blebbing relative to prototype IL477 LNPs.

[0189] DEFINITIONS

[0190]

[0096] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0191]

[0097] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, a pharmaceutical composition comprising lipid nanoparticles can be formulated for administration by parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration.PATENT

[0192] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0193]

[0098] Amino acid: In its broadest sense, as used herein, refers to any compound and / or substance that can be incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N–C(H)(RAA)–COOH, wherein RAAis an amino acid side chain. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides.

[0194] “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, PEGylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0195]

[0099] “Improve ” “increase”, “inhibit” or “reduce As used herein, the terms “improve”, “increase”, “inhibit”, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.PATENT

[0196] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0197]

[0100] Encapsulated: As used herein, the term “encapsulated” is used to refer to substances that are surrounded, contained within, enclosed, or encompassed, completely or in part, by another material. E.g., nucleic acid surrounded by lipid molecules, partially or completely shielding the nucleic acid from the external environment.

[0198]

[0101] Excipient: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0199]

[0102] Expression: As used herein, the term “expression” in relation to a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g, by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0200]

[0103] Gene: As used herein, the term “gene” refers to a nucleic acid sequence, such as DNA or RNA, that comprises partial length or entire length coding sequences required to produce a polypeptide or precursor polypeptide. In some embodiments, the term “gene product” refers to a product of a gene such as an RNA transcript or a polypeptide.

[0201]

[0104] Hydrodynamic diameter: As used herein, the term “hydrodynamic diameter” refers to the nanoparticle’s effective size in a fluid medium that accounts for the nanoparticle interactions with the solvent molecules around it (e.g., drag force exerted by the solvent on nanoparticle).

[0202]

[0105] Ionizable lipid: As used herein, the term “ionizable lipid” refers to a group of lipids that carry a net positive charge, such as at an acidic pH (e.g., pH of about 4.0), but remain neutrally charged at a physiological pH. The modulation of ionizability as a function of pH change is a factor in biocompatibility of the overall lipid nanoparticle. Ionizable lipids typically comprise of ionizable head group such as amine, a hydrophobic tail regionPATENT

[0203] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0204] comprising aliphatic chain of varying degree of saturation, and a linker connecting the two domains.

[0205]

[0106] Messenger RNA: As used herein, “messenger RNA” or “mRNA” refers to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’ -methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’-methoxy ribose residues, or a combination thereof. In general, mRNAs do not contain a substantial quantity of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues; or less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1% thymidine content). In some embodiments, an mRNA contains modified uridines at some or all of its uridine positions.

[0206]

[0107] Microfluidic Mixer: As used herein, the term “microfluidic mixer” refers to a device that enables rapid mixing in a highly controllable, reproducible manner that achieves homogeneous LNPs and high encapsulation efficiency. In these devices, individual streams of the ethanolic lipid mixture and aqueous oligonucleotide solution are rapidly combined. LNPs form as the two streams mix into a single chamber. Flow rate ratio and total flow rate can be altered to fine-tune LNPs.

[0207]

[0108] Substantially: The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0208]

[0109] T-junction Mixer or Y-junction Mixer: As used herein, the terms “T-junction mixer” or “Y-junction mixer” refers to a mixer where a T- or Y-connecter can be fitted with two inlets coupled to individual syringes containing the lipid mixture or oligonucleotide solution and one outlet to direct the LNPs into a collection chamber or tube. In some embodiments, the inlet flow rates can be controlled with syringe pumps.

[0209]

[0110] Nanoparticle: As used herein, the term “nanoparticle” refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticlePATENT

[0210] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0211] has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter from about 30 to about 170 nm. In some embodiments, lipid nanoparticles described herein can have an average hydrodynamic diameter that is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, lipid nanoparticles described herein have an average hydrodynamic diameter from between about 40 nm to about 100 nm. In some embodiments, lipid nanoparticles have an average hydrodynamic diameter of between about 50 nm and about 70 nm.

[0212]

[0111] Nanoparticle composition: As used herein, the term “nanoparticle composition” refers to a composition that contains at least one nanoparticle and at least one additional agent or ingredient. In some embodiments, a nanoparticle composition contains a substantially uniform collection of nanoparticles as described herein.

[0213]

[0112] Nucleic acid. As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in somePATENT

[0214] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0215] embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively, or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0216]

[0113] Particle size'. As used herein, “particle size” refers to average particle size or hydrodynamic diameter of a lipid nanoparticle. Particle size may be measured by Nanoparticle Tracking Analysis (NTA). Particle size is a parameter that may alter thePATENT

[0217] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0218] pharmacokinetics of an administered LNP. Smaller particles typically have longer circulation half-lives. Particles less than 100 nm can easily pass through fenestrated endothelium to penetrate target tissues. The particle size is dependent on the LNP preparation method. In some embodiments, extrusion can be used to achieve smaller, more uniform particle sizes.

[0219]

[0114] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and / or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0220]

[0115] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters.

[0221]

[0116] Pharmaceutically acceptable salt: As used herein, the term “pharmaceutically acceptable salt” refers to salts of such compounds that are appropriate for use in pharmaceutical contexts, i.e., salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J.

[0222] Pharmaceutical Sciences, 66: 1-19 (1977).

[0223]

[0117] Polydispersity index: As used herein, the term “poly dispersity index” or “PDF’ refers to a range of value between 0 to 1 that defines the homogeneity or heterogeneity of lipid nanoparticle population. PDI value close to 0 indicates homogeneity of lipidPATENT

[0224] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0225] nanoparticle population, whereas PDI value close to 1 indicates heterogeneity of the population. In some embodiments, the polydispersity index (PDI) is about 0.00. In some embodiments, the polydispersity index (PDI) is about 0.01. In some embodiments, the polydispersity index (PDI) is about 0.02. In some embodiments, the polydispersity index (PDI) is about 0.03. In some embodiments, the polydispersity index (PDI) is about 0.04. In some embodiments, the polydispersity index (PDI) is about 0.05. In some embodiments, the polydispersity index (PDI) is about 0.06. In some embodiments, the polydispersity index (PDI) is about 0.07. In some embodiments, the polydispersity index (PDI) is about 0.08. In some embodiments, the polydispersity index (PDI) is about 0.09. In some embodiments, the polydispersity index (PDI) is about 0.1.

[0226]

[0118] Polypeptide'. As used herein, the term “polypeptide” refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. As is known to those skilled in the art, polypeptides may be processed and / or modified.

[0227]

[0119] Protein'. As used herein, the term “protein” refers to one or more polypeptides that function as a discrete unit. If a single polypeptide is the discrete functioning unit and does not require permanent or temporary physical association with other polypeptides in order to form the discrete functioning unit, the terms “polypeptide” and “protein” may be used interchangeably. If the discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term “protein” refers to the multiple polypeptides that are physically coupled and function together as the discrete unit.

[0228]

[0120] Subject or Patient: As used herein, the term “subject” or “patient” refers to an individual suffering from a disease or disorder.

[0229]

[0121] Stability or Thermostability: As used herein, the term “stability” or “thermostability” refers to the minimal or negligible changes in the properties of lipid nanoparticles that would otherwise lead to degradation and / or decline in biological activity. The stability or thermostability of lipid nanoparticles is gauged by, among other properties, the particle size, uniformity, shape, and encapsulation.PATENT

[0230] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0231]

[0122] Therapeutically effective amount. As used herein, the term “therapeutically effective amount” means an amount effective, at dosages, frequency of administration and for duration of time necessary to achieve the desired results.

[0232] DETAILED DESCRIPTION

[0233]

[0123] The present disclosure provides, among other things, improved methods of making lipid nanoparticles that are stable, and efficacious, for example, for therapeutic use. The methods of the present invention provide, among other things, methods for making efficient stable lipid nanoparticles (LNPs) for gene editing, for example, encapsulating base editors and CRISPR Cas enzymes. In another aspect, provided herein are lipid nanoparticles made by the methods of the present invention.

[0234]

[0124] Typically, LNPs are prepared in an aqueous acidic buffer, providing a compact and stable lipid nanoparticle. Smaller particles typically have longer circulation halflives, as they evade elimination by the mononuclear phagocyte system (MPS) and have improved pharmacokinetics. Particles less than 100 nm can pass through fenestrated endothelium to penetrate target tissues. The size and uniformity of lipid nanoparticles may depend in part on the preparation method.

[0235]

[0125] Accordingly, in conventional methods of LNP preparation, typically LNPs are prepared by mixing an ethanolic lipid mixture with an acidic aqueous solution comprising nucleic acid cargo at around pH 4. Without wishing to be bound by any particular theory, it is contemplated that during particle formation, the ionizable lipids become positively charged, and through electrostatic interactions with the negatively charged phosphate backbone of nucleic acid polymers, promote incorporation of nucleic acid into the nanoparticle, yield a small compact stable lipid nanoparticle. The solution is subsequently dialyzed to yield LNPs at a neutral pH for storage and delivery.

[0236]

[0126] In one aspect, the inventors of the present invention found that mixing a first solution comprising one or more nucleic acids with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid to yield a mixed solution at a pH of between about 5.9 to 6.2 provided improved lipid nanoparticles encapsulating nucleic acid cargo, for example, for gene editing.

[0237]

[0127] Without wishing to be bound by any particular theory, it is contemplated that a conventional mixing method at a low pH to yield a mixed stable solution at about pH 4,PATENT

[0238] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0239] when subjected to subsequent dialysis, results in a pH shock, extensive lipid deprotonation and phase-separation leading to reduced LNP stability. By contrast, the higher pH mixing of the present invention causes a small pH shift upon dialysis associated with limited lipid deprotonation and phase separation resulting in more stable lipid nanoparticles by the improved methods of the present disclosure.

[0240]

[0128] By conventional methods, in order to ensure proper ionizable lipid protonation, the LNPs are prepared at an acidic pH (~4) which is significantly lower than the apparent pKa value of the ionizable lipids (typically ~ 6.5), followed by a buffer / pH exchange step (Albertson et al., C. H., Adv Drug Delivery Rev. 2022 Sep; 188: 114416).

[0241]

[0129] In one aspect, the inventors of the present invention found that mixing a first solution comprising one or more nucleic acids with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, provided lipid nanoparticles encapsulating nucleic acid cargo.

[0242]

[0130] In yet another aspect, the inventors of the present invention found that mixing a first solution comprising one or more nucleic acids with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid, wherein the method comprises adjusting the pH of the nucleic acid solution and / or the lipid solution to within 0.5 units of each other prior to mixing, followed by subsequent mixing carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, provided lipid nanoparticles encapsulating nucleic acid cargo.

[0243]

[0131] In another aspect, the present invention provides a method of preparing a lipid nanoparticle comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, wherein the first solution comprises one or more nucleic acids, and the second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid; wherein the mixing step is carried out to yield a mixed solution at a pH of between about 5.9 to 6.2, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo. In a further embodiment, the method further comprises a step of adjusting pH of the first solution and / or the second solution to within 0.5 units of each other prior to the mixing step. In a further embodiment, the pH of the first solution may be between pH 5.5 to pH 6.5 prior to the mixing step.PATENT

[0244] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0245]

[0132] In another aspect, the present invention provides a method of preparing a lipid nanoparticle comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, wherein the first solution comprises one or more nucleic acids, the second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid and a PEG lipid; wherein the mixing step is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo. In a further embodiment, the method further comprises a step of adjusting pH of the first solution and / or the second solution to within 0.5 units of each other prior to the mixing step.

[0246]

[0133] The LNP preparation methods of the present invention provide LNPs of improved size, homogeneity, poly dispersity, and encapsulation efficiency, which are stable and efficacious. In addition, stability is maintained, for example, after storage for between 1 to 6 months (e.g., at temperatures of about -20 °C, -80 °C), and after one or more cycles of freeze-thaw (e.g., 1, 2, 3 or more cycles). The present invention provides stable lipid nanoparticles with improved properties relative to lipid nanoparticles prepared by conventional methods leading to cost benefits, scalability, and batch-to-batch reproducibility in manufacturing, for example, for therapeutic use.

[0247]

[0134] In some embodiments, provided herein is a method, wherein the one or more nucleic acids comprise a guide RNA (gRNA) and an mRNA encoding a CRISPR base editor.

[0248] Exemplary Methods of Preparing Lipid Nanoparticles

[0249]

[0135] Provided herein are still further exemplary embodiments of methods of preparing lipid nanoparticles.

[0250]

[0136] Lipid nanoparticles encapsulating nucleic acid cargo can be prepared by mixing nucleic acid solution with a lipid solution in a two-stream mixing process, wherein one stream comprises an aqueous nucleic acid solution and the other stream comprises an ethanolic lipid solution. After mixing, the lipid nanoparticles are dialyzed into a solution at neutral pH for storage and administration.

[0251]

[0137] The present invention provides, among other things, a method of two-stream mixing whereby the pH of the mixed solution before dialysis is such that it yields lipid nanoparticles with improved, beneficial properties (e.g., encapsulation efficiency, poly dispersity, particle size) in the final product after dialysis. In some embodiments, thePATENT

[0252] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0253] method is a method of three or more stream mixing whereby the pH of the mixed solution before dialysis is such that it yields lipid nanoparticles with improved, beneficial properties (e.g., encapsulation efficiency, poly dispersity, particle size) in the final product after dialysis.

[0254]

[0138] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a nucleic acid cargo, the method comprising a product mixing step combining: a first solution comprising one or more nucleic acids with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid; wherein the product mixing step is carried out to yield a mixed solution at a pH of between about 5.9 to 6.2, thereby providing a product comprising a lipid particle encapsulating a nucleic acid cargo.

[0255]

[0139] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a nucleic acid cargo, the method comprising a product mixing step combining: a first solution comprising one or more nucleic acids with a second solution comprising at least an ionizable lipid with a other solution(s) comprising at least a phospholipid, a cholesterol lipid, or a PEG lipid; wherein the product mixing step is carried out to yield a mixed solution at a pH of between about 5.9 to 6.2, thereby providing a product comprising a lipid particle encapsulating a nucleic acid cargo.

[0256]

[0140] In some embodiments, provided herein are methods of preparing lipid nanoparticles by the methods provided herein. Also provided is a method of in vivo base editing comprising administering to a subject in need thereof, the LNPs described herein.

[0257]

[0141] In some embodiments, the lipid solution comprises an ionizable lipid in an aqueous buffer and / or organic solution. In some embodiments, the lipid nanoparticle solution may further comprise a buffering agent and / or a salt.

[0258]

[0142] In some embodiments, provided herein is a method, wherein the first solution (a) is an aqueous solution of water:ethanol in about a 3:1 (v / v) ratio. In some embodiments, the first solution (a) is an aqueous solution of water: ethanol in about a 4: 1 (v / v) ratio. In some embodiments, the first solution (a) is an aqueous solution of water: ethanol in about a 2:1 (v / v) ratio.

[0259]

[0143] In some embodiments, the nucleic acid solution comprises a buffering agent and / or a salt.

[0260]

[0144] Exemplary suitable buffering agents include, but are not limited to, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate,PATENT

[0261] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0262] sodium phosphate, HEPES, and the like. In some embodiments, the lipid solution and / or nucleic acid solution comprises a buffering agent at a concentration ranging from about 0.1-100 mM, from about 0.5-90 mM, from about 1.0-80 mM, from about 2-70 mM, from about 3-60 mM, from about 4-50 mM, from about 5-40 mM, from about 6-30 mM, from about 7-20 mM, from about 8-15 mM, from about 9-12 mM. In some embodiments, the lipid solution and / or nucleic acid solution comprises a buffering agent at a concentration of or greater than about 0.1 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. Exemplary suitable salts include, but are not limited to, potassium chloride, magnesium chloride, sodium chloride, and the like. In some embodiments, the lipid solution and / or nucleic acid solution comprises a salt at a concentration ranging from about 1-500 mM, from about 5-400 mM, from about 10- 350 mM, from about 15-300 mM, from about 20-250 mM, from about 30-200 mM, from about 40-190 mM, from about 50-180 mM, from about 50-170 mM, from about 50-160 mM, from about 50-150 mM, or from about 50-100 mM.

[0263]

[0145] In some embodiments, the lipid nanoparticle solution comprises a salt at a concentration of or greater than about 1 mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM.

[0264]

[0146] In some embodiments, the lipid solution and / or the nucleic acid solution has a pH ranging from about 5.0 to about 7.0, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5. In some embodiments, the lipid solution and / or the nucleic acid solution has a pH ranging from about 7.0 to about 8.0, about 7.1 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5. In some embodiments, a suitable lipid solution and / or nucleic acid solution may have a pH of or no greater than 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.

[0265]

[0147] In some embodiments, the lipid nanoparticle product solution has a pH ranging from about 5.0 to about 7.0, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.0 to about 6.9, about 6.0 to about 6.8, about 6.0 to about 6.7, about 6.0 to about 6.6, about 6.0 to about 6.5. In some embodiments, the lipid nanoparticle product solution has a pH ranging from about 7.0 to about 8.0, about 7.1 to about 7.8, about 7.2 to about 7.6, or about 7.3 to about 7.5. In some embodiments, a suitable lipid nanoparticle product solution has a pH of or no greater than 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0.PATENT

[0266] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0267]

[0148] In some embodiments, provided herein is a method, wherein the second solution (b) comprises between about 2% to 3% PEG. In some embodiments, the second solution (b) comprises about 2% PEG. In some embodiments, the second solution (b) comprises about 3% PEG.

[0268]

[0149] In some embodiments, provided herein is a method, wherein the product mixing step occurs in a T-junction or in a microfluidic mixer. In some embodiments, the product mixing step occurs in a T-junction. In some embodiments, the product mixing step occurs in a microfluidic mixer.

[0269]

[0150] In some embodiments, provided herein is a method, wherein the method comprises a buffer exchange step after the product mixing step.

[0270]

[0151] In some embodiments, provided herein is a method, wherein the method further comprises a concentration step after the buffer exchange step.

[0271]

[0152] In some embodiments, provided herein is a method, wherein the method further comprises a sterile filtration step, after a concentration step.

[0272]

[0153] In some embodiments, provided herein is a method, wherein the buffer exchange is carried out using dialysis and ultrafiltration, ultrafiltration and one or more dilution steps, or ultrafiltration and diafiltration. In some embodiments, the buffer exchange is carried out using dialysis and ultrafiltration. In some embodiments, the buffer exchange is carried out using ultrafiltration and one or more dilution steps. In some embodiments, the buffer exchange is carried out using ultrafiltration and diafiltration.

[0273]

[0154] In some embodiments, provided herein is a method, wherein the buffer exchange is carried out using Tangential Flow Filtration (TFF).

[0274]

[0155] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by no greater than about 1 nm to 5 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 1 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 2 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 3 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 4 nm after about threePATENT

[0275] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0276] rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 5 nm after about three rounds of freeze-thaw.

[0277]

[0156] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by no greater than about 6 nm to 10 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 6 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 7 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 8 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 9 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 10 nm after about three rounds of freezethaw.

[0278]

[0157] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by no greater than about 11 nm to 15 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 11 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 12 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 13 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 14 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 15 nm after about three rounds of freezethaw.

[0279]

[0158] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by no greater than about 16 nm to 20 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 16 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 17 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 18 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than aboutPATENT

[0280] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0281] 19 nm after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by no greater than about 20 nm after about three rounds of freezethaw.

[0282]

[0159] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by less than 50% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 40% after about one round of freeze-thaw. In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by less than 30% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 20% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 15% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 10% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 5% after about one round of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 1% after about one round of freeze-thaw.

[0283]

[0160] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by less than 50% after about two rounds of freezethaw. In some embodiments, the lipid nanoparticle product increases in size by less than 40% after about two rounds of freeze-thaw. In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by less than 30% after about two rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 20% after about two rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 15% after about two rounds of freezethaw. In some embodiments, the lipid nanoparticle product increases in size by less than 10% after about two rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 5% after about two rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 1% after about two rounds of freeze-thaw.

[0284]

[0161] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product increases in size by less than 50% after about three rounds of freezethaw. In some embodiments, the lipid nanoparticle product increases in size by less than 40% after about three rounds of freeze-thaw. In some embodiments, provided herein is a method,PATENT

[0285] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0286] wherein the lipid nanoparticle product increases in size by less than 30% after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 20% after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 15% after about three rounds of freezethaw. In some embodiments, the lipid nanoparticle product increases in size by less than 10% after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 5% after about three rounds of freeze-thaw. In some embodiments, the lipid nanoparticle product increases in size by less than 1% after about three rounds of freeze-thaw.

[0287]

[0162] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product is stable during storage at about -20 °C for at least about 1-6 months.

[0288]

[0163] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product is stable during storage at about -80 °C for at least about 1-6 months.

[0289]

[0164] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product is stored in Tris-buffered saline, Phosphate-buffered saline, HEPES or Tris-acetate buffer at pH 7.4. In some embodiments, the lipid nanoparticle product is stored in Tris-buffered saline at pH 7.4. In some embodiments, the lipid nanoparticle product is stored in phosphate-buffered saline at pH 7.4. In some embodiments, the lipid nanoparticle product is stored in HEPES at pH 7.4. In some embodiments, the lipid nanoparticle product is stored in Tris-acetate buffer at pH 7.4.

[0290]

[0165] In some embodiments, provided herein is a method, wherein the buffer further comprises sucrose.

[0291]

[0166] In some embodiments, provided herein is a method, wherein the lipid nanoparticle product is characterized by no substantial change in size, PDI, and / or activity following storage. In some embodiments, the lipid nanoparticle product is characterized by no substantial change in size following storage. In some embodiments, the lipid nanoparticle product is characterized by no substantial change in PDI following storage. In some embodiments, the lipid nanoparticle product is characterized by no substantial change in activity following storage.

[0292]

[0167] In some embodiments, the product mixing step is performed at a temperature of less than about 50 °C, less than about 45 °C, less than about 50 °C, less than about 35 °C, less than about 30 °C, less than about 28 °C, less than about 26 °C, less than about 24 °C, lessPATENT

[0293] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0294] than about 22 °C, less than about 25°C, less than about 20 °C, or less than about ambient temperature.

[0295]

[0168] In some embodiments, the product mixing step is performed at a temperature of about 50 °C, about 45 °C, about 40 °C, about 35 °C, about 30 °C, about 28 °C, about 26 °C, about 24 °C, about 22 °C, about 20 °C, or about ambient temperature.

[0296]

[0169] In some embodiments, the product mixing step is performed at a temperature of about 25 °C.

[0297]

[0170] In some embodiments, the product mixing step occurs in a T-junction or in a microfluidic mixer. In some embodiments, mixing processes use high energy mixers (e.g., T-junction, confined impinging jets, microfluidic mixers, vortex mixers) to introduce lipids (in ethanol) to a suitable anti-solvent (i.e., water) in a controllable fashion.

[0298]

[0171] Exemplary methods of using microfluidics to form lipid nanoparticles are described, for example, by Leung, A. K. K, et al., J Phys Chem, 116:18440-18450 (2012), Chen, D., et al., J Am Chem Soc, 134:6947-6951 (2012), and Belliveau, N. M., et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012), the disclosures of which are hereby incorporated by reference in their entireties. Briefly, a cargo, such as a cargo described herein, is prepared in a first buffer solution. The other lipid nanoparticle components (such as ionizable lipid, conjugate-linker lipids, cholesterol lipids, (e.g., cholesterol), and phospholipid) are prepared in a second buffer solution. In some embodiments, a syringe pump introduces the two solutions into a microfluidic device. The two solutions come into contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.

[0299]

[0172] In some embodiments, the method comprises a buffer exchange step after the product mixing step. In some embodiments, the method further comprises a concentration step after the buffer exchange step. In some embodiments, the method further comprises a sterile filtration step, after a concentration step. In some embodiments, the buffer exchange is carried out using dialysis and ultrafiltration, ultrafiltration and one or more dilution steps, or ultrafiltration and diafiltration. In some embodiments, the buffer exchange is carried out using Tangential Flow Filtration (TFF).

[0300]

[0173] In some embodiments, the lipid nanoparticle product increases in size by no greater than about 1-5 nm after about three rounds of freeze-thaw.PATENT

[0301] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0302]

[0174] In some embodiments, the lipid nanoparticle product is stable during storage at about -20 °C for at least about 1-6 months.

[0303]

[0175] In some embodiments, the lipid nanoparticle product is stable during storage at about -80 °C for at least about 1-6 months.

[0304]

[0176] In some embodiments, the lipid nanoparticle product is stored in Tris-buffered saline, Phosphate-buffered saline, HEPES or Tris-acetate buffer at pH 7.4.

[0305]

[0177] In some embodiments, the buffer further comprises sucrose.

[0306]

[0178] In some embodiments, the lipid nanoparticle product is characterized by no substantial change in size, PDI, and / or activity following storage.

[0307] pKa of Ionizable Lipids

[0308]

[0179] Ionizable lipids are protonated at low pH, which makes them positively charged, but since they remain non-ionized or neutral at physiological pH, they have fewer interactions with the anionic membranes of cells, providing advantages in delivery and improves biocompatibility of lipid nanoparticles. In the endosomes, where the pH is acidic, the ionizable lipids are protonated and become positively charged, promoting membrane stabilization and endosomal escape.

[0309]

[0180] In some aspects, the present disclosure provides a method of preparing lipid nanoparticles comprising a nucleic acid cargo, the method comprising a product mixing step combining:

[0310] a first solution (a) comprising one or more nucleic acids with a second solution (b) comprising an ionizable, a phospholipid, a cholesterol lipid, and a PEG lipid; wherein the mixing is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid,

[0311] thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0312]

[0181] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising one or more nucleic acids, with a second solution (b) comprising an ionizable lipid, a phospholipid, a cholesterol lipid and a PEG lipid; wherein the method comprises a step of adjusting pH of solution (a) and / or solution (b) to within 0.5 units of each other prior to the mixing, andPATENT

[0313] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0314] wherein the mixing is carried out at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0315]

[0182] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising one or more nucleic acids, with a second solution (b) comprising an ionizable lipid and one or more additional solutions comprising a PEG lipid, wherein the mixing is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0316]

[0183] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising RNA, with a second solution (b) comprising an ionizable lipid and one of more additional solutions comprising a phospholipid, a cholesterol lipid, or a PEG lipid, wherein the mixing is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0317]

[0184] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising one or more nucleic acids, with a second solution (b) comprising an ionizable lipid, a phospholipid, and a cholesterol lipid and one or more additional solutions comprising a phospholipid, a cholesterol lipid, or a PEG lipid; wherein the mixing is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0318]

[0185] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising one or more nucleic acids, with a second solution (b) comprising an ionizable lipid and a third solution (c) comprising a phospholipid, a cholesterol lipid, or a PEG lipid; wherein the mixing is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0319]

[0186] In some aspects, provided herein is a method of preparing lipid nanoparticles comprising a product mixing step combining: a first solution (a) comprising RNA, with a second solution (b) comprising an ionizable lipid and a third solution (c) comprising a phospholipid, a cholesterol lipid, or a PEG lipid, wherein the mixing is carried out to yield aPATENT

[0320] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0321] mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid, thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

[0322] Other Components of Lipid Nanoparticles

[0323]

[0187] In some aspects, the present invention provides for compositions, preparations, and / or nanomaterials that comprise lipid nanoparticles produced by the methods described herein. In some embodiments, lipid nanoparticles comprise one or more components such as an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid.

[0324]

[0188] In some embodiments, lipid nanoparticles comprise one or more compounds as described herein. In some embodiments, lipid nanoparticles comprise one or more ionizable lipids. In some embodiments, lipid nanoparticles comprise one or more phospholipids. In some embodiments, lipid nanoparticles comprise one or more cholesterol lipids. In some embodiments, lipid nanoparticles comprise one or more PEG lipids.

[0325] A. Ionizable lipids

[0326]

[0189] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that comprise one or more ionizable lipids. In some embodiments, an ionizable lipid may include an amine-containing group on the head group.

[0327]

[0190] In some embodiments, an ionizable lipid is present in a lipid nanoparticle (LNP) preparation from about 30 mole percent to about 70 mole percent of the total moles of lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 35 mole percent to about 65 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 40 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP.

[0328]

[0191] In some embodiments, an ionizable lipid is present from about 45 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 45.5 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 46 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 46.5 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP.PATENT

[0329] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0330]

[0192] In some embodiments, an ionizable lipid is present from about 47 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP.

[0331]

[0193] In some embodiments, an ionizable lipid is present from about 50 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 59.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 59 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 58.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 58 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 57.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 57 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 56.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 56 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 51 mole percent to about 55.5 mole percent, based on total moles of the lipids present in the LNP.

[0332]

[0194] In some embodiments, an ionizable lipid is present from about 51 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP.

[0333]

[0195] In some embodiments, an ionizable lipid is present from about 50 mole percent to about 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54 mole percent to about 57 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54 mole percent to about 56.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54 mole percent to about 56 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54PATENT

[0334] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0335] mole percent to about 55.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54 mole percent to about 55 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present from about 54 mole percent to about 54.5 mole percent, based on total moles of the lipids present in the LNP.

[0336]

[0196] In some embodiments, an ionizable lipid is present from about 54 mole percent to about 57 mole percent, based on total moles of the lipids present in the LNP.

[0337]

[0197] In some embodiments, an ionizable lipid is present at about 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present at about 47.5 mole percent, based on total moles of the lipids present in the LNP.

[0338]

[0198] In some embodiments, an ionizable lipid is present at about 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, or 60 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, an ionizable lipid is present at about 60 mole percent, based on total moles of the lipids present in the LNP.

[0339]

[0199] In various exemplary embodiments, an ionizable lipid is any ionizable lipid known in the art. As non-limiting examples, an ionizable lipid is (25)-2,5- bis(3 -aminopropylamino)- 7V-[2-(dioctadecylamino)acetyl] pentanamide (DOGS; Transfectam), 7V1 - [2-(( 15)- 1 - [(3 -aminopropyl)amino] -4- [di (3 - aminopropyl)amino] butylcarboxamido)ethyl]-3,4- difoleyloxy]- benzamide (MVL5), DC-cholesterol and N4-cholesterylspermine (GL67), 1,2- dilinoleyloxy- N, N- dimethyl-3- aminopropane (DLin-DMA), 2,2- dilinoleyl-4- dimethylaminoethyl-[l,3]- dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31- tetraen-19- yl 4-(dimethylamino) butanoate (DLin-MC3-DMA; MC3), CAS Registry No. 1857340-78-3 (4A3-SC8), CAS Registry No.

[0340] 1857341-90-2 (5A2SC8), CAS Registry No. 2768211-13-6 (4A3-Cit), CAS Registry No. 2412492-09-0 (A18-Iso5-2DC18), CAS Registry No. 2639634-80-1 (C14-4), 8-[(2-hydroxyethyl)[8-(nonyloxy)-8-oxooctyl]amino]-octanoic acid, 1 -octylnonyl ester (lipid 5), di(dec-3-yn-l-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (lipid A6), 4-(dimethylamino)-butanoic acid, 2-[di-(9Z,12Z)-9,12-octadecadien-l-ylamino]ethyl ester (lipid 8), tetrakis(8-methylnonyl) 3,3',3",3"'-(((methylazanediyl)bis(propane-3,l-PATENT

[0341] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0342] diyl))bis(azanetriyl))tetrapropionate (306Oi10), tetrakis(2-(octyldisulfaneyl)ethyl) 3,3',3",3"'-(((methylazanediyl)Z»z5(propane-3,l-diyl))Z»z5(azanetriyl))tetrapropionate (306-O12B), 3,3',3",3"'-(((methylazanediyl)Zh (propane-3,l-diyl))Zh (azanetriyl))te / ra XN-(2-(dodecyldisulfaneyl)ethyl)propanamide) (306-N16B), N-[(25Z)-14-[(9Z)-9-octadecen-l-yloxy]-3,6,9,12,16-pentaoxatetratriacont-25-en-l-yl]-lH-imidazole-5-carboxamide (DOG-IM4), 4-[[(9Z)-l-oxo-9-octadecen-l-yl]oxy]-benzeneacetic acid, 1, 1 '-[dithiobis(2, 1-ethanediyl-l,4-piperidinediyl-2,l -ethanediyl)] ester (ssPalmO-Phe), l,l'-[[2-[4-[2-[[2-[ > Z5(2-hydroxydodecyl)amino]ethyl](2-hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]Z»z5-2-dodecanol (C 12-200), 1,1'-[[2-[2-[4-[2-[[2-[2-[Z> Z5(2-hydroxytetradecyl)amino]ethoxy]ethyl](2-hydroxytetradecyl)amino]ethyl]-l-piperazinyl]ethoxy]ethyl]imino]Z>z -2-tetradecanol (C 14-4), 1, 1', 1", 1 '"-[1,4-piperazinediylbis(3,l-propanediylnitrilo)]tetrakis-2-dodecanol (246C10), 3,6-Z>zs[4-[Z»zX2-hydroxydodecyl)amino]butyl]-2,5-piperazinedione (cKK-E12), 3,6-Z>z [4-[Z>z [(9Z, 12Z)-2-hydroxy-9,12-octadecadien-l-yl]amino]butyl]-2,5-piperazinedione (OF-02), 9Z,12Z-octadecadienoic acid, 1, 1', 1", 1 '''-[(3,6-dioxo-2,5-piperazinediyl)Z>z (4, 1 -butanediylnitrilodi- 2.1 -ethanediyl)] ester (OF-Deg-Lin), 9,12-octadecadienoic acid, (9Z,12Z)-l,r,l",l'"-[(3,6-dioxo-2,5-piperazinediyl)Z»z5(4,l-butanediylnitrilodi-4,l-butanediyl)] ester (OF-C4-Deg-Lin), 2-(((4-(dimethylamino)butanoyl)oxy)methyl)-2-((((Z)-tetradec-9-enoyl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)-Z>z (tetradec-9-enoate) (TCL053), [(4-hydroxybutyl)azanediyl]di(hexane- 6.1-diyl) bis(2 -hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 9Z,12Z-octadecadienoic acid, 3-[4,4-A / .s(octyloxy)-l-oxobutoxy]-2-[[[[3-(diethylamino)propoxy]carbonyl]oxy]methyl]propyl ester (LP-01), N1,N3,N5-[3-(didodecylamino)propyl]-1,3,5-benzenetricarboxamide (TT3), 4,4'-[[[[3-(dimethylamino)propyl]thio]carbonyl]imino]Z>z -butanoic acid, l,l'- > Z5(l-heptyloctyl) ester (ATX-100), 8-[[8-[(l-ethylnonyl)oxy]-8-oxooctyl][3-[[2-(methylamino)-3,4-dioxo-l-cyclobuten-l-yl]amino]propyl]amino]-octanoic acid, 1-octylnonyl ester (Lipid 29), among others.

[0343]

[0200] Other exemplary, non-limiting ionizable lipids include those described in International Publication Nos. WO 2021 / 021634A1; WO 2021 / 113365A1; WO 2021 / 141969A1; WO 2022 / 140239 Al; WO 2022 / 140252A1; WO 2022 / 140238 Al; WO 2022 / 159421A1; WO 2022 / 159472A1; WO 2022 / 159475 Al; WO 2022 / 159463 Al; WO 2023 / 121965A1; WO 2023 / 121964A1; WO 2023 / 121971A1; WO 2023 / 121970A1; WOPATENT

[0344] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0345] 2023 / 121975A1; and WO 2024 / 019936A1; each of which is incorporated by reference in its entirety.

[0346]

[0201] Exemplary, non-limiting ionizable lipids include those described in International Publication No. WO 2015 / 095340A1, which is incorporated by reference in its entirety.

[0347]

[0202] Additional exemplary, non-limiting ionizable lipids include those described in International Publication Nos. WO 2023 / 114943 Al; WO 2023 / 114937A1; WO 2023 / 114944A1; WO 2022 / 016070A1; WO 2020 / 146805 Al; WO 2020 / 081938 Al; WO 2019 / 036030A1; WO 2019 / 036000 Al; WO 2019 / 036028A1; WO 2019 / 036008A1; WO 2018 / 200943 Al; WO 2018 / 081480A1; WO 2017 / 117528A1; WO 2017 / 075531 Al; WO 2017 / 004143A1; and WO 2015 / 199952A1; each of which is incorporated by reference in its entirety.

[0348]

[0203] Additional exemplary, non-limiting ionizable lipids include those described in International Publication Nos. WO 2023 / 107669A1; WO 2022 / 204288 Al; WO 2021 / 055849A1; WO 2021 / 055835A1; WO 2021 / 055833A1; WO 2020 / 061367 Al; WO 2018 / 232120A1; WO 2018 / 170306A1; WO 2017 / 112865A1; WO 2017 / 049245 A3; each of which is incorporated by reference in its entirety.

[0349]

[0204] Still further exemplary, non-limiting ionizable lipids include those described in International Publication Nos. WO 2023 / 198857A1; WO 2023 / 178167A1; WO 2022 / 066916A1; WO 2022 / 066678 Al; WO 2021 / 202694 Al; WO 2020 / 257611 Al; WO 2020 / 257716A1; WO 2020 / 243540A1; WO 2020 / 227085 Al; WO 2020 / 219427 Al; WO 2020 / 214946A1; WO 2020 / 097384A1; WO 2019 / 232095 Al; WO 2019 / 232097 Al; WO 2019 / 232208 Al; WO 2019 / 226925 Al; WO 2019 / 222424 Al; WO 2015 / 184256A3; WO 2013 / 149140A1; and WO 2012 / 170889A8; each of which is incorporated by reference in its entirety.

[0350]

[0205] Other exemplary, non-limiting ionizable lipids include those described in International Publication Nos. WO 2020 / 219876A1; WO 2020 / 118041 Al; and WO 2020 / 072605 Al; each of which is incorporated by reference in its entirety.

[0351]

[0206] Still other exemplary, non-limiting ionizable lipids include those described in International Publications Nos. WO 2020 / 072324A1; WO 2013 / 086373 Al; WO

[0352] 2013 / 086354A1; WO 2013 / 086322A1; WO 2011 / 153493 A3; WO 2010 / 054405 Al; each of which is incorporated by reference in its entirety.PATENT

[0353] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0354]

[0207] Among other things, in some embodiments, a lipid nanoparticle composition comprises an ionizable lipid. In some embodiments, a lipid nanoparticle preparation comprises an ionizable lipid; a phospholipid; a PEG-lipid; and a cholesterol lipid (e.g., cholesterol). In some embodiments, an ionizable lipid is or comprises a structure according to a compound described herein. In some embodiments, an ionizable lipid is present in a LNP preparation from about 30 mole percent to about 70 mole percent, based on total moles of components of the lipid nanoparticle.

[0355]

[0208] In various exemplary embodiments, an ionizable lipid is selected from the group consisting of IL1 through IL492 as depicted in the table below.

[0356] Table 1. Ionizable lipids IL1 to IL492,

[0357]

[0358] PATENT

[0359] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0360]

[0361] IL10,PATENT

[0362] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0363] IL12,

[0364] IL15,

[0365]

[0366] PATENT

[0367] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0368] IL18,

[0369]

[0370] IL21,PATENT

[0371] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0372] IL23,

[0373]

[0374] IL27,PATENT

[0375] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0376] IL30,

[0377]

[0378] PATENT

[0379] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0380] IL33,

[0381]

[0382] IL37,PATENT

[0383] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0384] IL39,

[0385] IL42,

[0386]

[0387] PATENT

[0388] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0389] IL44

[0390] IL47, IL48, IL49,

[0391]

[0392] PATENT

[0393] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0394] IL54,

[0395] IL56,

[0396] IL58,

[0397] IL60,

[0398] IL62,

[0399]

[0400] PATENT

[0401] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0402] IL69,

[0403]

[0404] IL79,PATENT

[0405] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0406] IL81,

[0407] IL82,

[0408] IL85,

[0409] IL86,

[0410] IL89,

[0411]

[0412] IL90, IL91,PATENT

[0413] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0414] IL95, IL99, IL101,

[0415]

[0416] IL104, IL105,PATENT

[0417] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0418] IL111,

[0419] IL114, IL116,

[0420]

[0421] PATENT

[0422] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0423] IL120

[0424] IL122

[0425] IL124

[0426] IL126

[0427] IL128

[0428] IL131

[0429] HO

[0430]

[0431] PATENT

[0432] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0433] IL1135,

[0434] IL139,

[0435]

[0436] PATENT

[0437] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0438] IL153,

[0439] IL155,

[0440] IL159,

[0441]

[0442] IL161,PATENT

[0443] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0444] IL163,

[0445]

[0446] PATENT

[0447] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0448] IL179,

[0449] IL183,

[0450] IL187,

[0451]

[0452] PATENT

[0453] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0454] IL196

[0455] IL199 IL201

[0456]

[0457] IL203PATENT

[0458] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0459] IL207, IL209,

[0460]

[0461] IL216, IL217,PATENT

[0462] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0463] IL218, IL219,

[0464] IL220, IL221,

[0465] drb

[0466] IL229,

[0467]

[0468] PATENT

[0469] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0470] IL233,

[0471] IL240

[0472]

[0473] IL242 IL243PATENT

[0474] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0475] IL247,

[0476] IL253,

[0477]

[0478] PATENT

[0479] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0480] IL265, IL267,

[0481]

[0482] IL268, IL269,PATENT

[0483] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0484] IL272,

[0485] IL275,

[0486] IL281,

[0487]

[0488] PATENT

[0489] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0490] IL282, IL283,

[0491] IL287, IL291,

[0492]

[0493] IL294, IL295,PATENT

[0494] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0495] IL301, IL303, IL304, IL306, IL307,

[0496]

[0497] IL308, IL309,PATENT

[0498] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0499] IL313,

[0500] IL316,

[0501] IL317,

[0502] IL321,

[0503]

[0504] PATENT

[0505] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0506] IL324, IL325, IL326,

[0507] IL330,

[0508] IL334,

[0509]

[0510] IL336,PATENT

[0511] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0512] IL341, IL342, IL344,

[0513]

[0514] IL348,PATENT

[0515] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0516]

[0517] PATENT

[0518] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0519] IL358

[0520] IL363

[0521] IL364

[0522] HO

[0523] HO

[0524]

[0525] PATENT

[0526] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0527] IL374, IL376,

[0528] IL380, IL381,

[0529]

[0530] IL385,PATENT

[0531] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0532] IL387,

[0533] IL388,

[0534] IL390, IL391,

[0535] IL395,

[0536]

[0537] IL397,PATENT

[0538] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0539] IL398,

[0540]

[0541] IL407,PATENT ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0542] o

[0543] IL416,

[0544] IL418, o-

[0545]

[0546] PATENT

[0547] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0548] IL420,

[0549] IL424, o IL427,

[0550]

[0551] IL429, IL430,PATENT

[0552] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0553] IL435, IL436, o IL437,

[0554]

[0555] IL439,PATENT

[0556] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0557] IL441,

[0558] IL442,

[0559] IL443,

[0560] IL444,

[0561]

[0562] PATENT

[0563] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0564] IL446,

[0565] IL447,

[0566] IL448,

[0567] IL449,

[0568]

[0569] IL450,PATENT

[0570] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0571] IL451,

[0572] IL452,

[0573] IL453,

[0574] IL454,

[0575]

[0576] IL455,PATENT

[0577] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0578]

[0579] PATENT

[0580] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0581]

[0582] PATENT

[0583] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0584]

[0585] PATENT

[0586] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0587] IL474, IL475, O IL476, 0

[0588]

[0589] IL477,PATENT

[0590] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0591] IL478,

[0592] IL479,

[0593]

[0594] PATENT

[0595] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0596] IL484,

[0597] IL487,

[0598] IL488,

[0599]

[0600] PATENT

[0601] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0602]

[0603] or a pharmaceutically acceptable salt thereof.

[0604]

[0209] In some embodiments, an ionizable lipid is selected from the group consisting of IL7, IL45, IL76, and IL477 as depicted in the table below.

[0605] Table 2. Ionizable lipids IL7, IL45, IL76, and IL466

[0606]

[0607] PATENT

[0608] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0609] (X X3 o

[0610] o (

[0611]

[0612] IL7, or a pharmaceutically acceptable salt thereof.

[0613]

[0210] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.

[0614]

[0211] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a13C- or14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

[0615] B. Cholesterol lipid or Sterol lipid

[0616]

[0212] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that comprise one or more cholesterol lipids as described herein.

[0617]

[0213] In some embodiments, a cholesterol lipid is a cholesterol, or a variant or derivative thereof. In some embodiments, a cholesterol is modified. In some embodiments, aPATENT

[0618] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0619] cholesterol is an oxidized cholesterol. In some embodiments, a cholesterol is esterified cholesterol. Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized. In some embodiments, a cholesterol can be oxidized on the betaring structure or on the hydrocarbon tail structure. In some embodiments, a cholesterol is a phytosterol. Exemplary cholesterols that are considered for use in the disclosed lipid nanoparticles include but are not limited to 25-hydroxycholesterol (25-OH), 20a-hydroxycholesterol (20a-OH), 27-hydroxycholesterol, 6-keto-5a-hydroxycholesterol, 7-ketocholesterol, 7P-hydroxycholesterol, 7a-hydroxycholesterol, 7P-25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, a cholesterol is an unmodified cholesterol.

[0620]

[0214] In some embodiments, a cholesterol lipid is present in a lipid nanoparticle (LNP) preparation from about 20 mole percent to about 50 mole percent of the total moles of lipids present in the LNP. In some embodiments, a cholesterol lipid is present from about 25 mole percent to about 45 mole percent, based on total moles of the lipids present in the LNP.

[0621]

[0215] In some embodiments, a cholesterol lipid is present from about 30 mole percent to about 40 mole percent, based on total moles of the lipids present in the LNP.

[0622]

[0216] In some embodiments, a cholesterol lipid is present at about 30, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mole percent based on total moles of the lipids present in the LNP. In some embodiments, a cholesterol lipid is present at about 35 mole percent based on total moles of the lipids present in the LNP. In some embodiments, a cholesterol lipid is present at about 40 mole percent based on total moles of the lipids present in the LNP.

[0623] C. PEG lipids

[0624]

[0217] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that comprise one or more polyethylene glycol (PEG) lipids as described herein. PEG lipids can also include PEG-modified lipids, examples of which are described herein.

[0625]

[0218] PEG or PEG-modified lipids may be alternately referred to as PEGylated lipids or PEG-lipids. Inclusion of a PEGylating lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, the PEGylation is reversible in that the PEG moiety is gradually released in blood circulation.PATENT

[0626] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0627] Exemplary PEG-lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20. PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof may also be suitable. For example, in some embodiments, a PEG lipid may be PEG(2000)-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE lipid.

[0628]

[0219] In some embodiments, the PEG lipid comprises PEG-c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG-DSPE, or any combination thereof. In some embodiments, the PEG lipid comprises a PEG-dimyristylglycerol (PEG-DMG) conjugate.

[0629]

[0220] In some embodiments, a PEG lipid comprises DiMystyrlGlycerol (PEG-DMG), 1,2-Dipalmitoyl-rac-glycerol, methoxypolyethylene Glycol (DPG-PEG), or 1,2-Distearoyl-rac-glycero-3 -methylpolyoxyethylene (PEG-DSG). In some embodiments, a conjugate-linker lipid has an average molecular mass from about 500 Da to about 5000 Da. In some embodiments, a conjugate-linker lipid has an average molecular mass of about 2000 Da.

[0630]

[0221] In some embodiments, a PEG lipid is present from about 0.1 mole percent to about 5 mole percent, based on total moles of the lipids present in the lipid nanoparticle (LNP). In some embodiments, a PEG lipid is present from about 1 mole percent to about 5 mole percent, based on total moles of the lipids present in the LNP.

[0631]

[0222] In some embodiments, a PEG lipid is present from about 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5, 4.75, or 5 mol percent, based on total moles of the lipids present in the LNP. In some embodiments, a PEG lipid is present at about, 2.5 mole percent, based on total moles of the lipids present in the LNP.

[0632]

[0223] In some embodiments, a PEG lipid is present from about 2 mole percent to about 3 mole percent, based on total moles of the lipids present in the LNP.

[0633]

[0224] In some embodiments, a PEG lipid is present at about 2, 2.25, 2.5, 2.75, or 3 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, a PEG lipid is present at about, 2.5 mole percent, based on total moles of the lipids present in the LNP.PATENT

[0634] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0635] D. Phospholipids

[0636]

[0225] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that comprise one or more phospholipids as described herein. In some embodiments, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that comprise one or more (poly)unsaturated lipids.

[0637]

[0226] In some embodiments, one or more phospholipids include a phospholipid moiety. In some embodiments, one or more phospholipids include one or more fatty acid moieties. In some embodiments, one or more phospholipids include a phospholipid moiety and one or more fatty acid moieties. In some embodiments, a phospholipid moiety includes but is not limited to phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and sphingomyelin. In some embodiments, a fatty acid moiety includes but is not limited to lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alphalinolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Nonnatural species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid may be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0638]

[0227] Exemplary phospholipids include but are not limited to 1,2-distearoyl-snglycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-PATENT

[0639] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0640] cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In some embodiments, a phospholipid is DSPC. In some embodiments, a phospholipid is DMPC.

[0641]

[0228] In some embodiments, the phospholipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

[0642]

[0229] In some embodiments, the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), or a combination thereof.

[0643]

[0230] In some embodiments, the phospholipid comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0644]

[0231] In some embodiments, the phospholipid comprises 1,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC).

[0645]

[0232] In some embodiments, a phospholipid is present from about 1 mole percent to about 15 mole percent, based on total moles of the lipids present in the lipid nanoparticlePATENT

[0646] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0647] (LNP). In some embodiments, a phospholipid is present from about 5 mole percent to about 12 mole percent, based on total moles of the lipids present in the LNP.

[0648]

[0233] In some embodiments, a phospholipid is present from about 7 mole percent to about 10 mole percent, based on total moles of the lipids present in the LNP.

[0649]

[0234] In some embodiments, a phospholipid is present at about 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75 or 10 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, a phospholipid is present at about 7.5 mole percent, based on total moles of the lipids present in the LNP. In some embodiments, a phospholipid is present at about 9.5 mole percent, based on total moles of the lipids present in the LNP.

[0650] Features of Lipid Nanoparticles

[0651]

[0235] The lipid nanoparticles made by the improved methods of the present invention have various beneficial properties for functional activity, e.g., particle size, poly dispersity, and encapsulation efficiency, among others.

[0652] E. Particle size

[0653]

[0236] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that have an average particle size or hydrodynamic diameter from about 30 to about 220 nm. In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter that is about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, or 220 nm, or any discrete quantity therebetween. For example, in some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein have an average hydrodynamic diameter from between about 40 nm to about 200 nm. In some embodiments, the preparations, nanoparticles, and / or nanomaterials have an average hydrodynamic diameter from about 40 nm to about 100 nm. In some embodiments, the preparations, nanoparticles, and / or nanomaterials have an average hydrodynamic diameter from about 50 nm to about 70 nm.

[0654]

[0237] In some embodiments, the lipid nanoparticle size is between about 50 nm to 120 nm. In some embodiments, lipid nanoparticles described herein have an average particlePATENT

[0655] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0656] size or hydrodynamic diameter from about 50 nm to about 120 nm, or any discrete quantity therebetween. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter that is about 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, or 120 nm or any discrete quantity therebetween.

[0657]

[0238] In some embodiments, the lipid nanoparticle size is between about 40 nm to about 100 nm. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter from about 40 nm to about 100 nm, or any discrete quantity therebetween. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter from about 50 nm to about 70 nm, or any discrete quantity therebetween. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter that is about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, or 100 nm, or any discrete quantity therebetween.

[0658]

[0239] In some embodiments, the lipid nanoparticle size is between about 50 nm to about 70 nm. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter from about 50 nm to about 70 nm, or any discrete quantity therebetween. In some embodiments, lipid nanoparticles described herein have an average particle size or hydrodynamic diameter that is about 50 nm, 55 nm, 60 nm, 65 nm, or 70 nm, or any discrete quantity therebetween.

[0659]

[0240] In some embodiments, size is measured by Nanoparticle Tracking Analysis (NTA). NTA utilizes light scattering and Brownian motion in order to obtain the nanoparticle size distribution of samples in liquid suspension. Briefly, particles in liquid suspension are loaded into a sample chamber, which is illuminated by a specially shaped laser beam.

[0660] Particles in the path of the beam scatter the laser light which is easily collected by the 20x microscope objective and is viewed with a digital camera. The camera captures a video of the particles moving under Brownian motion. The Nanoparticle Tracking Analysis (NTA) software then analyses many particles individually and simultaneously (particle-by-particle), and by using the Stokes Einstein equation, calculates their hydrodynamic diameters or particle sizes. As used herein, “hydrodynamic diameter” and “particle size” are used interchangeably.PATENT

[0661] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0662] F. Polydispersity

[0663]

[0241] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials that have a polydispersity index (PDI) of about 0.01 to about 0.3. In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, or any range having endpoints defined by any two of the aforementioned values. For example, in some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09. In some embodiments, the lipid nanoparticle has a polydispersity index of 0.05.

[0664]

[0242] In some embodiments, lipid nanoparticles described herein have a PDI from about 0.01 to about 0.3. In some embodiments, lipid nanoparticles described herein have a PDI that is about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, or 0.3, or any discrete intervening quantity therebetween. For example, in some embodiments, lipid nanoparticles described herein have a PDI from about 0.05 to about 0.2, about 0.06 to about 0.1, or about 0.07 to about 0.09. In some embodiments, the PDI of the lipid nanoparticle product is about 0.1. In some embodiments, the PDI of the lipid nanoparticle product is about 0.05.

[0665]

[0243] In some embodiments, provided herein is a method, wherein the

[0666] polydispersity index (PDI) of the lipid nanoparticle product is less than about 0.1. In some embodiments, the PDI of the lipid nanoparticle product is 0.01, 0.02, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, or 0.09 or any discrete intervening quantity therebetween. In some embodiments, the polydispersity is about 0.01. In some embodiments, the polydispersity is about 0.02. In some embodiments, the polydispersity is about 0.03. In some embodiments, the polydispersity is about 0.04. In some embodiments, the poly dispersity is about 0.05. In some embodiments, the poly dispersity is about 0.06. In some embodiments, the

[0667] poly dispersity is about 0.07. In some embodiments, the poly dispersity is about 0.08. In some embodiments, the poly dispersity is about 0.09.

[0668]

[0244] In some embodiments, the polydispersity index (PDI) of the lipid nanoparticle product is less than about 0.05. In some embodiments, the PDI of the lipid nanoparticle product is 0.00, 0.01, 0.02, 0.03, 0.04 or any discrete intervening quantity therebetween. In some embodiments, the polydispersity is about 0.01. In some embodiments,PATENT

[0669] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0670] the polydispersity is about 0.02. In some embodiments, the polydispersity is about 0.03. In some embodiments, the polydispersity is about 0.04.

[0671]

[0245] In some embodiments, PDI is measured by Dynamic Light Scattering (DLA). Poly dispersity or dispersity refers to the degree of “non-uniformity” of a distribution. Using DLA, the size distribution of particles is measured. In DLA, native distribution is the light intensity distribution which indicates how much light is scattered from the various size “slices” or “bins” of lipid nanoparticles. The mean size and the standard deviation from the mean is obtained from the statistics of the distribution. The (absolute) standard deviation (or “halfwidth”) of the distribution is compared to the mean, and a relative polydispersity, i.e., standard deviation relative to mean is obtained. Overall polydispersity index PDI is the square of the light scattering polydispersity. A PDI of 0.0 indicates a perfectly uniform sample. Accordingly, samples with greater homogeneity have a low PDI, e.g., 0.01 to 1 or 0.01 to 0.05.

[0672] G. Encapsulation efficiency

[0673]

[0246] Among other things, the present disclosure describes compositions, preparations, nanoparticles, and / or nanomaterials, wherein encapsulation efficiency of provided compositions, preparations, nanoparticles, and / or nanomaterials is from about 80% to about 100%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and / or nanomaterials described herein is about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100%, or any discrete intervening quantity therebetween.

[0674]

[0247] In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is at least about 90%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 90%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 91%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 92%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 93%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 94%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 95%.

[0675]

[0248] In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is at least about 95%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 96%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 97%. In some embodiments, the encapsulation efficiencyPATENT

[0676] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0677] of the lipid nanoparticle product is about 98%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 99%. In some embodiments, the encapsulation efficiency of the lipid nanoparticle product is about 100%.

[0678]

[0249] For example, in some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and / or nanomaterials described herein is from about 90% to about 100%, about 95% to about 100%, about 95% to about 98%, or about 95.5% to about 97.5%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and / or nanomaterials described herein is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, encapsulation efficiency of compositions, preparations, nanoparticles, and / or nanomaterials described herein is about 100%.

[0679] H. N: P ratio

[0680]

[0250] As used herein, “N: P ratio” or “N / P ratio” refers to the ionizable lipid nitrogen: nucleic acid phosphate (N: P) molar ratio which represents the charge balance between the cationic tertiary amine of the ionizable cationic lipid and the anionic phosphate group in the nucleotide polymer backbone. Based on this property, ionizable lipids form a complex with nucleotides. In some embodiments, provided herein is a method, wherein the lipid particle product comprises an N:P ratio of between about 10:1 to about 1:1. In some embodiments, LNPs have a N:P ratio of about 10:1. In some embodiments, LNPs have a N:P ratio of about 9:1. In some embodiments, LNPs have a N: P ratio of about 8: 1. In some embodiments, LNPs have a N:P ratio of about 7:1. In some embodiments, LNPs have a N:P ratio of about 6:1. In some embodiments, LNPs have aN: P ratio of about 5:1. In some embodiments, LNPs have aN: P ratio of about 4:1. In some embodiments, LNPs have aN: P ratio of about 3:1. In some embodiments, LNPs have a N: P ratio of about 2: 1. In some embodiments, LNPs have a N:P ratio of about 5:1. In some embodiments, LNPs have a N:P ratio of about 1:1.

[0681] I. Stability

[0682]

[0251] LNPs provided herein showed improved stability, for example, after storage for a period of time. In some embodiments, the LNPs are stable for at least 1 week. In some embodiments, the LNPs are stable for at least 2 weeks. In some embodiments, the LNPs are stable for at least 3 weeks. In some embodiments, the LNPs are stable for at least 5 weeks. In some embodiments, the LNPs are stable for at least 1 month. In some embodiments, the LNPs are stable for at least 2 months. In some embodiments, the LNPs are stable for at least 3PATENT

[0683] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0684] months. In some embodiments, the LNPs are stable for at least 4 months. In some embodiments, the LNPs are stable for at least 5 months. In some embodiments, the LNPs are stable for at least 6 months. In some embodiments, the LNPs are stable for at least 7 months. In some embodiments, the LNPs are stable for at least 8 months. In some embodiments, the LNPs are stable for at least 9 months. In some embodiments, the LNPs are stable for at least 10 months. In some embodiments, the LNPs are stable for at least 11 months. In some embodiments, the LNPs are stable for at least 1 year. In some embodiments, the LNPs are stable for about 1 year or more.

[0685]

[0252] In some embodiments, the stability of the LNPs is increased by at least 1 week compared to controls prepared by conventional methods. In some embodiments, the stability of the LNPs is increased by at least 2 weeks compared to controls. In some embodiments, the stability of the LNPs is increased by at least 3 weeks compared to controls. In some embodiments, the stability of the LNPs is increased by at least 4 weeks compared to controls. In some embodiments, the stability of the LNPs is increased by at least 1 month compared to controls. In some embodiments, the stability of the LNPs is increased by at least 2 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 3 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 4 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 5 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 6 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 7 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 8 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 9 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 10 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 11 months compared to controls. In some embodiments, the stability of the LNPs is increased by at least 1 year compared to controls. In some embodiments, the stability of the LNPs is increased by 1 year or more compared to controls.

[0686] °C

[0687]

[0253] In some embodiments, the LNPs are stable at -20 for at least 1 week. In some embodiments, the LNPs are stable at -20 °C for at least 2 weeks. In some embodiments, the LNPs are stable at -20 °C for at least 3 weeks. In some embodiments, the LNPs are stable at -20 °C for at least 4 weeks. In some embodiments, the LNPs are stable at -20 °C for 1 month. In some embodiments, the LNPs are stable at -20 °C for at least 2 months. In somePATENT

[0688] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0689] embodiments, the LNPs are stable at -20 °C for at least 3 months. In some embodiments, the LNPs are stable at -20 °C for at least 4 months. In some embodiments, the LNPs are stable at -20 °C for at least 5 months. In some embodiments, the LNPs are stable at -20 °C for at least 6 months. In some embodiments, the LNPs are stable at -20 °C for at least 7 months. In some embodiments, the LNPs are stable at -20 °C for at least 8 months. In some embodiments, the LNPs are stable at -20 °C for at least 9 months. In some embodiments, the LNPs are stable at -20 °C for at least 10 months. In some embodiments, the LNPs are stable at -20 °C for at least 11 months. In some embodiments, the LNPs are stable at -20 °C for at least 1 year. In some embodiments, the LNPs are stable at -20 °C for more than 1 year.

[0690] °C

[0691]

[0254] In some embodiments, the LNPs are stable at -80 for at least 1 week. In some embodiments, the LNPs are stable at -80 °C for at least 2 weeks. In some embodiments, the LNPs are stable at -80 °C for at least 3 weeks. In some embodiments, the LNPs are stable at -80 °C for at least 4 weeks. In some embodiments, the LNPs are stable at -80 °C for 1 month. In some embodiments, the LNPs are stable at -80 °C for at least 2 months. In some embodiments, the LNPs are stable at -80 °C for at least 3 months. In some embodiments, the LNPs are stable at -80 °C for at least 4 months. In some embodiments, the LNPs are stable at -80 °C for at least 5 months. In some embodiments, the LNPs are stable at -80 °C for at least 6 months. In some embodiments, the LNPs are stable at -80 °C for at least 7 months. In some embodiments, the LNPs are stable at -80 °C for at least 8 months. In some embodiments, the LNPs are stable at -80 °C for at least 9 months. In some embodiments, the LNPs are stable at -80 °C for at least 10 months. In some embodiments, the LNPs are stable at -80 °C for at least 11 months. In some embodiments, the LNPs are stable at -80 °C for at least 1 year. In some embodiments, the LNPs are stable at -80 °C for more than 1 year.

[0692]

[0255] In some embodiments, the LNPs made of the methods of the present invention demonstrate a negligible reduction in hydrodynamic diameter after storage at -20°C for up to 4 weeks (e.g., less than about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, including all values and ranges therebetween) (e.g., as compared to a control, e.g. LNPs made by conventional methods).

[0693]

[0256] In some embodiments, the LNPs made of the methods of the present invention demonstrate a negligible increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks (e.g., an increase of less than about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,PATENT

[0694] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0695] 9%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, including all values and ranges therebetween) (e.g., as compared to a control, e.g. LNPs made by conventional methods).

[0696]

[0257] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 20% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 19% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 18% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 17% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 16% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks.

[0697]

[0258] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 15% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 14% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 13% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 12% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 11% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks.

[0698]

[0259] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 10% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 9% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 8% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 7% increase in hydrodynamic diameter after storage at -20°CPATENT

[0699] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0700] for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 6% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks.

[0701]

[0260] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 5% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 4% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 3% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 2% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 1% increase in hydrodynamic diameter after storage at -20°C for up to 4 weeks.

[0702]

[0261] In some embodiments, the LNPs made of the methods of the present invention demonstrate a negligible increase in hydrodynamic diameter after storage at -80°C for up to 3 months (e.g., an increase of less than about a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40% or 50%, including all values and ranges therebetween) (e.g., as compared to a control, e.g. LNPs made by conventional methods).

[0703]

[0262] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 20% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 19% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 18% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 17% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 16% increase in hydrodynamic diameter after storage at -80°C for up to 3 months.

[0704]

[0263] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 15% increase in hydrodynamic diameter after storage at -PATENT

[0705] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0706] 80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 14% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 13% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 12% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 11% increase in hydrodynamic diameter after storage at -80°C for up to 3 months.

[0707]

[0264] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 10% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 9% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 8% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 7% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 6% increase in hydrodynamic diameter after storage at -80°C for up to 3 months.

[0708]

[0265] In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 5% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 4% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 3% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 2% increase in hydrodynamic diameter after storage at -80°C for up to 3 months. In some embodiments, the LNPs made of the methods of the present invention demonstrate less than 1% increase in hydrodynamic diameter after storage at -80°C for up to 3 months.PATENT

[0709] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0710]

[0266] In some embodiments, the LNPs made of the methods of the present invention demonstrate a negligible reduction in pharmacological or biological activity (e.g., less than about a 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40% or 50%, including all values and ranges therebetween, reduction in the biological or pharmacological activity of an encapsulated polynucleotide) (e.g., as compared to a control, e.g. LNPs made by conventional methods).

[0711]

[0267] In some embodiments, LNPs described herein provides comparable stability as compared to an equivalent LNP produced by conventional methods. In some embodiments, the stability does not appreciably change during storage of an LNP, including any exemplary, storage duration, and / or temperature described herein. In some embodiments, the stability of an LNP does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40% or 50%, including all values and ranges therebetween (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), following storage, including according to any exemplary storage duration, and / or temperature as described herein.

[0712]

[0268] In some embodiments, LNPs described herein provides comparable stability as compared to LNPs produced by conventional methods. In some embodiments, the stability of LNPs does not change by more than about 1%, 2%, 2.5%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 18.5%, 20%, 25%, 30%, 35%, 40% or 50% (e.g., no more than about 30%, 25%, 20%, 15%, 10%, or 5%), including all values and ranges therebetween compared to LNPs produced by any conventional methods.

[0713] J. Nucleic Acid Cargo

[0714]

[0269] In some embodiments, provided herein is a lipid nanoparticle (LNP) composition prepared by a method of the present invention. In some embodiments, provided herein is an LNP, wherein the one or more nucleic acids is a messenger RNA (mRNA), a guide RNA (gRNA), a circular RNA (circDNA), or a DNA. In some embodiments, the one or more nucleic acids is a mRNA. In some embodiments, the one or more nucleic acids is a gRNA. In some embodiments, the one or more nucleic acids is a circular RNA. In some embodiments, the one or more nucleic acids is a DNA.

[0715]

[0270] A person skilled in the art would appreciate that double stranded nucleic acids (e.g., siRNA and plasmid DNA) exhibit different physiochemical properties than singlePATENT

[0716] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0717] stranded nucleotides (e.g., mRNA). Moreover, it is well-recognized that LNPs that are used to encapsulate small nucleic acids may not be readily adaptable to large sequences (e.g., in excess of 3000 nucleotides). The present disclosure provides LNPs that are surprisingly effective at encapsulating and delivering large nucleic acid molecules. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises between about 3000 to 10,000 nucleotides (nts). In some embodiments, the nucleic acid comprises between about 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000 nucleotides, or any discrete intervening quantity therebetween. For example, in some embodiments, the nucleic acid comprising between about 4000 to 7500 nucleotides or about 4500 to 7000 nucleotides. In some embodiments, the nucleic acid comprises between about 3000 to 8000 nucleotides. In some embodiments, the nucleic acid comprises between about 5000 to 7000 nucleotides.

[0718]

[0271] In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 3000 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 3500 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 4000 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 4500 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 5000 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 5500 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 6000 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 6500 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 7000 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 7500 nucleotides. In some embodiments, provided herein is an LNP, wherein the nucleic acid comprises about 8000 nucleotides.

[0719]

[0272] In some embodiments, provided herein is an LNP, wherein the one or more nucleic acids encodes a base editor.

[0720]

[0273] In some embodiments, provided herein is an LNP, wherein the LNP further encapsulates a small molecule or protein. In some embodiments, the LNP further encapsulates a small molecule. In some embodiments, the LNP further encapsulates a protein.

[0721]

[0274] In some embodiments, provided herein is a method of in vivo base editing comprising administering to a subject in need thereof, the LNP described herein.PATENT

[0722] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0723]

[0275] In some embodiments, provided herein is a method, wherein about 20%-60% base editing is achieved. In some embodiments, about 20% base editing is achieved or at least 20% base editing is achieved. In some embodiments, about 25% base editing is achieved or at least 25% base editing is achieved. In some embodiments, about 30% base editing is achieved or at least 30% base editing is achieved. In some embodiments, about 35% base editing is achieved or at least 35% base editing is achieved. In some embodiments, about 40% base editing is achieved or at least 40% base editing is achieved. In some embodiments, about 45% base editing is achieved or at least 45% base editing is achieved. In some embodiments, about 50% base editing is achieved or at least 50% base editing is achieved. In some embodiments, about 55% base editing is achieved or at least 55% base editing is achieved. In some embodiments, about 60% base editing is achieved or at least 60% base editing is achieved.

[0724]

[0276] In some embodiments, provided herein is a method, wherein at least 50% base editing is achieved.

[0725]

[0277] Cargo delivered via a LNP preparation may be a biologically active agent. In some embodiments, the cargo is or comprises one or more biologically active agents, such as mRNA, guide RNA (gRNA), nucleic acid, RNA-guided DNA-binding agent, expression vector, template nucleic acid, antibody (e.g., monoclonal, chimeric, humanized, nanobody, and fragments thereof etc.), cholesterol, hormone, peptide, protein, chemotherapeutic and other types of antineoplastic agent, low molecular weight drug, vitamin, co-factor, nucleoside, nucleotide, oligonucleotide, enzymatic nucleic acid, antisense nucleic acid, triplex forming oligonucleotide, antisense DNA or RNA composition, chimeric DNA: RNA composition, allozyme, aptamer, ribozyme, decoys and analogs thereof, plasmid (e.g., circular DNA or circular RNA) and other types of vectors, and small nucleic acid molecule, RNAi agent, short interfering nucleic acid (siNA), short interfering RNA (siRNA), doublestranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA) and “selfreplicating RNA” (encoding a replicase enzyme activity and capable of directing its own replication or amplification in vivo) molecules, peptide nucleic acid (PNA), a locked nucleic acid ribonucleotide (LNA), morpholino nucleotide, threose nucleic acid (TNA), glycol nucleic acid (GNA), sisiRNA (small internally segmented interfering RNA), and iRNA (asymmetrical interfering RNA). The above list of biologically active agents is exemplary only, and is not intended to be limiting. Such compounds may be purified or partially purified, and may be naturally occurring or synthetic, and may be chemically modified.PATENT

[0726] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0727]

[0278] In some embodiments, cargo delivered via a LNP preparation is an RNA, such as an mRNA molecule encoding a protein of interest. For example, in some embodiments, an mRNA for expressing a protein such as green fluorescent protein (GFP), an RNA-guided DNA-binding agent, or a Cas nuclease is described herein. LNP preparations that include a Cas nuclease mRNA, for example a Class 2 Cas nuclease mRNA that allows for expression in a cell of a Class 2 Cas nuclease such as a Cas9 or Cpfl protein are provided. Further, cargo may contain one or more guide RNAs or nucleic acids encoding guide RNAs. A template nucleic acid, e.g., for repair or recombination, may also be included in the composition or a template nucleic acid may be used in the methods described herein. In some embodiments, cargo comprises an mRNA encoding any Cas9 and optionally any corresponding gRNA known in the art. In some embodiments, cargo comprises an mRNA that encodes a Streptococcus pyogenes Cas9, optionally and an S. pyogenes gRNA. In some embodiments, cargo comprises an mRNA that encodes Neisseria meningitidis Cas9, optionally and an nme gRNA.

[0728] K. CRISPR / Cas Cargo

[0729]

[0279] In some embodiments, a lipid nanoparticle composition is prepared by a method described herein comprising a nucleic acid cargo. In some embodiments, the one or more nucleic acids is a mRNA, a gRNA, a circular RNA, or a DNA. In some embodiments, the one or more nucleic acids encodes a base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the LNP further encapsulates a small molecule or protein.

[0730]

[0280] In some embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise an mRNA encoding an RNA-guided DNA-binding agent, such as a Cas nuclease. In particular embodiments, the disclosed compositions, preparations, nanoparticles, and / or nanomaterials comprise an mRNA encoding a Class 2 Cas nuclease, such as S. pyogenes Cas9.

[0731]

[0281] As used herein, an “RNA-guided DNA binding agent” refers to a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA binding agents include Cas cleavage enzymes / nickases and inactivated forms thereof (“dCas DNA binding agents”).PATENT

[0732] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0733]

[0282] As used herein, the term “Cas nuclease” encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. Cas cleavases / nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas1O, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. As used herein, a “Class 2 Cas nuclease” is a single chain polypeptide with RNA-guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases (e.g., H840A, D10A, or N863 A variants), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpfl, C2cl, C2c2, C2c3, HF Cas9 (e.g., N497A, R661 A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(l.l) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpfl protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpfl sequences of Zetsche are incorporated by reference in their entirety herein. See, e.g., Zetsche, Tables SI and S3. See, e.g, Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015), the contents of which are hereby incorporated in its entirety herein.

[0734]

[0283] As used herein, the term “ribonucleoprotein”, “RNP” or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.

[0735]

[0284] In some embodiments, cargo for a LNP preparation includes at least one guide RNA comprising guide sequences that direct an RNA-guided DNA binding agent, which can be a nuclease (e.g., a Cas nuclease such as Cas9), to a target DNA. gRNA may guide the Cas nuclease or Class 2 Cas nuclease to a target sequence on a target nucleic acid molecule. In some embodiments, a gRNA binds with and provides specificity of cleavage by a Class 2 Cas nuclease. In some embodiments, a gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex such as a CRISPR / Cas9 complex. In some embodiments, a CRISPR / Cas complex may be a Type-II CRISPR / Cas9 complex. In some embodiments, a CRISPR / Cas complex may be a Type-V CRISPR / Cas complex, suchPATENT

[0736] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0737] as a Cpfl / guide RNA complex. Cas nucleases and cognate gRNAs may be paired. gRNA scaffold structures that pair with each Class 2 Cas nuclease vary with the specific CRISPR / Cas system.

[0738]

[0285] Guide RNA”, “gRNA”, and simply “guide” are used herein interchangeably to refer to either a crRNA (also known as CRISPR RNA), or the combination of a crRNA and a trRNA (also known as tracrRNA). Guide RNAs can include modified RNAs as described herein. The crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or in two separate RNA molecules (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. trRNA may be a naturally occurring sequence, or a trRNA sequence with modifications or variations compared to naturally occurring sequences.

[0739] Pharmaceutical Compositions and Administration Methods

[0740]

[0286] In some embodiments, provided herein is a method of in vivo base editing comprising administering to a subject in need thereof, a LNP described herein. In some embodiments, about 20%-60% base editing is achieved. In some embodiments, base editing of about 20%, 30%, 40%, 50%, 60%, or any discrete intervening quantity therebetween is achieved. In some embodiments, at least 50% base editing is achieved.

[0741]

[0287] In some embodiments, provided herein is a method of treating a disease, the method comprising administering a therapeutically effective amount of a lipid nanoparticle composition made by methods described herein to a subject in need thereof.

[0742]

[0288] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

[0743]

[0289] In some embodiments, pharmaceutical compositions may include one or more nanoparticle compositions described herein. For example, a pharmaceutical composition may comprise one or more nanoparticle compositions including one or more different therapeutic and / or prophylactics including but not limited to one or more nucleic acids of different types or encode different agents. In some embodiments, a pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or accessory ingredients including but not limited to a pharmaceutically acceptable carrier.PATENT

[0744] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0745]

[0290] A pharmaceutical composition may be administered to a subject. In some embodiments, a pharmaceutical composition is administered as described herein. In some in vivo approaches, the nanoparticle compositions disclosed herein are administered to a subject in a therapeutically effective amount as described herein.

[0746]

[0291] In some embodiments, the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to devise an appropriate dosage level and dosing regimen using the pharmaceutical compositions described herein for treatment of various conditions in various patients. For example, in some embodiments, a selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. In some embodiments, generally dosage levels of about 0.001 mg to about 5 mg of nucleic acid per kg of body weight are administered each dosage to mammals. More specifically, in some embodiments, a preferential dose for nucleic acids within the disclosed nanoparticles is about 0.01 mg / kg to about 1.0 mg / kg.

[0747]

[0292] In some embodiments, a pharmaceutical composition described herein is administered locally, for example by injection directly into a site to be treated. Typically, the injection causes an increased localized concentration of the composition which is greater than that which can be achieved by systemic administration. In some embodiments, a pharmaceutical composition described herein can be combined with a matrix as described herein to assist in creating an increased localized concentration of the polypeptide compositions by reducing the passive diffusion of the polypeptides out of the site to be treated.

[0748]

[0293] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein, including those containing lipid nanoparticles, are administered in an aqueous solution, by parenteral injection. In some embodiments, a preparation may also be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of a lipid nanoparticle, and optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions optionally include one or more for the following: diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HC1, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., TWEEN 20 (polysorbate-20), TWEEN 80 (polysorbate-80)), anti-PATENT

[0749] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0750] oxidants (e.g., ascorbic acid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and redissolved / resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions.

[0751]

[0294] In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are administered in controlled release formulations. In some embodiments, controlled release polymeric devices are made for long term release systemically following implantation of a polymeric device (such as a rod, cylinder, film, disk) or injection (such as microparticles). In some embodiments, a matrix is in the form of microparticles such as microspheres. In some embodiments, an agent is dispersed within a solid polymeric matrix or microcapsules. In some embodiments, a core is of a different material than a polymeric shell of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, a peptide is dispersed or suspended in a core, which may be liquid or solid in nature, of any of the described compositions, preparations, nanoparticles, and / or nanomaterials. Unless specifically defined herein, microparticles, microspheres, and microcapsules are used interchangeably. In some embodiments, a polymer may be cast as a thin slab or film, ranging from nanometers to four centimeters, a powder produced by grinding or other standard techniques, or even a gel such as a hydrogel.

[0752]

[0295] In some embodiments, non-biodegradable matrices are used for delivery of the described compositions, preparations, nanoparticles, and / or nanomaterials. In some embodiments, biodegradable matrices are used for delivery of the described compositions, preparations, nanoparticles, and / or nanomaterials.

[0753]

[0296] In some embodiments, biodegradable matrices comprise natural or synthetic polymers. In some embodiments, synthetic polymers provide improved characterization of degradation and release profiles. In some embodiments, a polymer is selected based on the period over which release is desired. In some embodiments, linear release is employed. In some embodiments, a pulse release or “bulk release” is used for effective results. In somePATENT

[0754] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0755] embodiments, a polymer is in the form of a hydrogel (typically in absorbing up to about 90% by weight of water). In some embodiments, the polymer is crosslinked with multivalent ions or polymers.

[0756]

[0297] The matrices are formed by solvent evaporation, spray drying, solvent extraction and other methods known to those skilled in the art. Bioerodible microspheres are prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowitz and Langer, J. Controlled Release, 5:13-22 (1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988), the disclosure of which is hereby incorporated by reference in its entirety herein.

[0757]

[0298] In some embodiments, the described compositions, preparations, nanoparticles, and / or nanomaterials are formulated for local release to treat the area of implantation or injection - which will typically deliver a dosage that is much less than the dosage for treatment of an entire body - or systemic delivery. These can be implanted or injected subcutaneously, into the muscle, fat, or administered orally.

[0758]

[0299] Among other things, the present invention provides for compositions, preparations, nanoparticles, and / or nanomaterials that comprise cargo as described herein. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials include a therapeutic or prophylactic agent for delivery to a subject. In some embodiments, a therapeutic or prophylactic agent is encapsulated by a lipid nanoparticle. In some embodiments, a lipid nanoparticle is loaded with one or more nucleic acids.

[0759] Methods of use

[0760]

[0300] Among other things, the present disclosure describes methods of using compositions, preparations, nanoparticles, and / or nanomaterials described herein. For example, in some embodiments, the present disclosure describes methods of using compositions, preparations, nanoparticles, and / or nanomaterials to deliver cargo to specific cells, tissues, or organs, as described herein. As another example, in some embodiments, the present disclosure describes methods of treatment and / or delaying and / or arresting progression of a disease or disorder using compositions, preparations, nanoparticles, and / or nanomaterials as described herein. In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein are for use in medicine.PATENT

[0761] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0762]

[0301] In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials described herein deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials deliver therapeutic or prophylactic agents to specific cells or organs in a subject in need thereof in the absence of a targeting ligand. In some embodiments, the compositions, preparations, nanoparticles, and / or nanomaterials are useful to treat or prevent diseases in a subject in need thereof.

[0763] Methods of delivering cargo to cells, tissue, or organs

[0764]

[0302] Among other things, in some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein target a particular type or class of cells (e.g., cells of a particular organ or system thereof), tissues, and / organs. In some embodiments, the present disclosure provides methods of delivering one or more cargos described herein to a subject in need thereof. In some embodiments, such methods comprise in vivo and / or in vitro delivery. In some embodiments, such methods comprise in vivo delivery. In some embodiments, such methods comprise in vitro delivery. In some embodiments, the present disclosure provides for methods of delivering one or more therapeutic and / or prophylactic nucleic acids to a subject in need thereof are described herein.

[0765]

[0303] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to liver cells in the subject. Exemplary liver cells include but are not limited to hepatocytes.

[0766]

[0304] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to spleen cells in the subject. Exemplary spleen cells include but are not limited to splenic monocytes, splenic T cells, splenic memory B cells, or splenic B cells.

[0767]

[0305] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to bone marrow cells in the subject. Exemplary bone marrow cells include but are not limited to bone marrow monocytes, bone marrow B cells, bone marrow memory B cells, or bone marrow T cells.

[0768]

[0306] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specificallyPATENT

[0769] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0770] delivered to immune cells in the subject. Exemplary immune cells include but are not limited to CD8+, CD4+, or CD8+CD4+ cells.

[0771]

[0307] In some embodiments, a composition, preparation, nanoparticle, and / or nanomaterial comprises a therapeutic and / or prophylactic of interest that may be specifically delivered to hematopoietic stem cells in the subject. Unless otherwise specified, it is understood that the terms “hematopoietic stem cells (HSCs)” and “hematopoietic stem and progenitor cells (HSPCs)” are used interchangeably in the present disclosure.

[0772]

[0308] In some embodiments, the lipid nanoparticles can be formulated to be delivered in the absence of a targeting ligand to mammalian liver hepatocytes, liver immune cells, spleen T cells, or lung endothelial cells. Specific delivery to a particular class or type of cells indicates that a higher proportion of lipid nanoparticles are delivered to target type or class of cells. In some embodiments, specific delivery may result in a greater than 2-fold, 5-fold, 10-fold, 15-fold, or 20-fold compared to delivery using a conventional nanoparticle system (e.g., MC3 -containing LNPs).

[0773] Methods of producing a polypeptide

[0774]

[0309] Among other things, in some embodiments, methods of using compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used for methods of producing a polypeptide. Among other things, in some embodiments, lipid nanoparticles described herein can be used for producing a polypeptide in a target cell in a subject in need thereof. For example, in some embodiments, lipid nanoparticles described herein can be used for producing a polypeptide in a target cell in a subject in need thereof. In some embodiments, compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein comprise one or more nucleic sequences to be delivered to a cell.

[0775]

[0310] In some embodiments, one or more nucleic acids are expressed in a cell. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.PATENT

[0776] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0777] Methods of gene regulation

[0778]

[0311] Among other things, in some embodiments, methods of using compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein are used for gene regulation. Among other things, in some embodiments, lipid nanoparticles described herein can be used for reducing and / or increasing gene expression in a target cell in a subject in need thereof. For example, in some embodiments, lipid nanoparticles described herein can deliver one or more nucleic acids to a target cell in the subject without a targeting ligand. In some embodiments, a nucleic acid is an inhibitor nucleic acid. In some embodiments, an inhibitory nucleic acid is an siRNA. In some embodiments, a nucleic acid is a nucleic acid described herein. As another example, in some embodiments, lipid nanoparticles described herein can deliver cargo to a target cell in the subject without a targeting ligand. In some embodiments, cargo is any cargo described herein.

[0779]

[0312] Among other things, in some embodiments, methods of using compositions, preparations, nanoparticles, and / or nanomaterials disclosed herein for editing of a gene in a cell in a subject in need thereof.

[0780]

[0313] In some embodiments, a cell that is targeted for gene regulation is an immune cell. The immune cell can be a T cell, such as CD8+ T cell, CD4+ T cell, or T regulatory cell. Other exemplary immune cells for gene editing include but are not limited to macrophages, dendritic cells, B cells or natural killer cells. In some embodiments, the cell that is targeted for gene regulation in a hepatocyte.

[0781]

[0314] Exemplary genes that can be targeted include but are not limited to T cell receptors, B cell receptors, CTLA4, PD1, FOXO1, FOXO3, AKTs, CCR5, CXCR4, LAG3, TIM3, Killer immunoglobulin-like receptors, GITR, BTLA, LFA-4, T4, LFA-1, Bp35, CD27L receptor, TNFRSF8, TNFRSF5, CD47, CD52, ICAM-1, LFA-3, L-selectin, Ki-24, MB1, B7, B70, M-CSFR, TNFR-II, IL-7R, OX-40, CD137, CD137L, CD30L, CD40L, FasL, TRAIL, CD257, LIGHT, TRAIL-R1, TRAILR2, TRAIL-R4, TWEAK -R, TNFR, BCMA, B7DC, BTLA, B7-H1, B7-H2, B7-H3, ICOS, VEGFR2, NKG2D, JAG1, GITR, CD4, CCR2, GATA-3, MTORC1, MTORC2, RAPTOR, GATOR, FOXP3, NF AT, IL2R, and IL7. Other exemplary genes that can be targeted include but are not limited to OCT, G6Pase, Mut, PCCA, PCCB, PCSK9, ALAS1, and PAH. Exemplary tumor-associated antigens that can be recognized by T cells and are contemplated for targeting, include but are not limited to MAGE1, MAGE3, MAGE6, BAGE, GAGE, NYESO-1, MART1 / Melan A, MC1R, GP100,PATENT

[0782] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0783] tyrosinase, TRP-1, TRP-2, PSA, CEA, Cyp-B, Her2 / Neu, hTERT, MUC1, PRAME, WT1, RAS, CDK-4, MUM-1, KRAS, MSLN and β-catenin.

[0784] Subjects to be treated.

[0785]

[0315] In some embodiments, subjects who are treated are mammals experiencing cancer, autoimmune disease, infections disease, organ transplant, organ failure, protein deficiency, or a combination thereof. In some embodiments, a subject is a human. In some embodiments, methods described herein may cause hepatocytes to translate certain proteins. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a hepatocyte. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic T cell. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic B cell. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a splenic monocyte. In some embodiments, methods described herein may be used to deliver one or more DNA, mRNA, sgRNA, or siRNA to a bone marrow cell.

[0786]

[0316] It should be understood that the order of steps or order for performing certain action is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0787]

[0317] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0788] EXAMPLES

[0789]

[0318] Various aspects of the invention are described in further detail in the following examples. The following examples describe some of the preferred modes of making and practicing the present invention. However, it should be understood that thesePATENT

[0790] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0791] examples are for illustrative purposes only and are not meant to limit the scope of the invention.

[0792] Table 3. IL7, IL45, IL76, and IL466

[0793] Ionizable Name Source

[0794] Lipid

[0795] IL76 ((3 -hydroxypropyl)azanediyl)bis(heptane-7, 1 -diyl) Example 8-4 in

[0796] bis(4,4-bis(((Z)-oct-5-en-l-yl)oxy)butanoate) International Publication WO 2022 / 140252

[0797] IL477 l-(3-((4,4-bis(((Z)-oct-5-en-l-yl)oxy)butanoyl)oxy)-2- Example 4-71 in

[0798] (((((1 -ethylpiperidin-3 -yl)methoxy)carbonyl)oxy) International methyl)propyl) 7-(3 -pentyloctyl) heptanedioate Publication WO 2022 / 159472

[0799] IL45 3-((4,4-bis(((Z)-oct-5-en-l-yl)oxy)butanoyl)oxy)-2- Example 7-49 in

[0800] (((7-((2- International butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4- Publication WO ((4-(pyrrolidin- 1 -yl)butanoyl)oxy)decanoate 2024 / 019936

[0801] IL7 3-((4,4-bis(((Z)-oct-5-en-l-yl)oxy)butanoyl)oxy)-2- Example 7-7 in

[0802] (((7-((2- International butyloctanoyl)oxy)heptanoyl)oxy)methyl)propyl 4- Publication WO

[0803]

[0804] (((2-(pyrrolidin-l-yl)ethyl)carbamoyl)oxy)decanoate 2024 / 019936

[0805] General Two-Stream LNP Preparation Protocol

[0806]

[0319] Unless otherwise noted, LNPs were formed through the rapid mixing of an aqueous RNA stream and ethanolic lipid stream at a flow rate ratio of 3: 1, in a T-mixer as outlined in FIG. 1. The flow was driven by syringe pumps. Freshly formed LNP solution, which contained 25% ethanol, was collected in a sterile container prior to dialysis. For consistency of LNP quality, the first 3 seconds and last 2 seconds of solution from T-mixer was discarded due to potential non-steady-state flow rates.

[0807]

[0320] The RNA packet was prepared by mixing RNA stock (dissolved in water), ultrapure water, and a 100 mM citrate buffer stock. Default concentrations in the RNA packet: [total RNA] = 0.2 mg / mL; [mRNA] / [sgRNA] = 1 (mass ratio); [Citrate] = 50 mM. For consistency, mixing pH refers to the pH of the 100 mM citrate buffer stock (+ / - 0.1 pH units) prior to addition of RNA and water.

[0808]

[0321] The lipid packet contains Ionizable lipid (IL) such as any one of the ionizable lipids in Tables 1, 2, or 3, cholesterol (Chol), DSPC, and PEG-lipid in 100% ethanol. Lipid concentrations are such to achieve target LNP composition using indicated flow rates.PATENT

[0809] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0810]

[0322] Within about 10 minutes of formation, the freshly formed LNP solution was buffer exchanged against >30x volume excess of Tris-NaCl buffer (50 mM Tris, 150 mM NaCl, pH 7.4) using appropriately sized dialysis cassettes (Slide-A-Lyzer G2; 20,000 MWCO; Thermo Scientific). To ensure proper buffer exchange, the LNPs were dialyzed for 12-18 hours with one or two buffer changes. For small scale formulations, the initial dialysis was done for 1 hour at room temperature and the final dialysis was done at 4 °C overnight after another buffer change. For large scale formulations, the initial dialysis was done for 2 hours at room temperature, followed by a buffer change and 2 hours at 4°C. The final dialysis was done at 4°C overnight after another buffer change.

[0811]

[0323] After dialysis, the LNP solution was transferred to ultra-centrifugation filters (Amicon Ultra 100K MWCO; Millipore Sigma) and concentrated up to about 1.5 mg / mL by centrifugation at 2000g.

[0812]

[0324] The concentrated LNPs were sterile filtered using 0.2 pm syringe filter(s) (0.2 pm Acrodisc syringe filter, Pall Corporation) in a biological safety cabinet.

[0813] Freezing and thawing of LNPs for stability and dosing Protocol

[0814]

[0325] LNPs were first dialyzed and concentrated to 0.5-1.5 mg / mL (by total RNA) in 50 mM Tris, 150 mM NaCl at pH 7.4, filtered (using 0.22 mm syringe filter), then diluted in 40% Sucrose solution in water at a 3: 1 volumetric ratio. The resulting LNP buffer composition became 37.5 mM Tris, 112.5 mM NaCl and 10% Sucrose. These buffered LNPs were aliquoted and stored frozen at -80 °C until further use. Aliquot volume was 0.05 - 2.0 mL for all test articles. For stability evaluation, some aliquots were stored at -20°C. To prepare dosing solution, frozen LNP stock was first allowed to completely thaw at room temperature. The LNP stock was then diluted with pure water and TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4) to achieve the desired LNP concentration and tonicity near 300 mOsm / kg.

[0815] General Sample Preparation for Cryoelectron Microscopy

[0816]

[0326] Cryo-electron microscopy images of LNPs mentioned herein were prepared from frozen LNP samples (50-100 pL aliquots) with known RNA concentrations and IxTBSPATENT

[0817] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0818] buffer for dilution if the imaging operator deemed necessary. Images were then analyzed and interpreted for LNP morphology.

[0819] Example 1. Stability and Potency of LNPs produced by the improved two-stream process comprising IL7

[0820]

[0327] This example illustrates size, encapsulation efficiency and stability, and delivery potency in wild type mice (balb / C) for nineteen compositional variants of IL7. The LNPs were produced at a mixing pH of 4 or 6 by the general two-stream LNP preparation protocol described above. Briefly, a first solution (a) comprising of a chemically modified sgRNA and an mRNA encoding an adenine base editor with 50 mM or 25 mM citrate concentration and 0.1 or 0.2 mg / mL total RNA concentration was mixed with a second solution (b) comprising IL7, cholesterol, DSPC, and DMG-PEG lipid to yield a mixed product solution at pH 4 to pH 6.4. One flow rate was utilized during mixing, namely, 60 mL / min.

[0821]

[0328] Each LNP was measured via Dynamic Light Scattering (DLS) and Ribogreen Assay to measure particle size, total RNA concentration and encapsulation before and after freezing 50 pL aliquots by the General Freezing and thawing LNPs for Stability.

[0822]

[0329] Compositional variants were prepped for dosing solutions by the Freezing and Thawing of LNPs for dosing Protocol. Fresh particles, as in never frozen, were diluted with pure water and TBS buffer (50 mM Tris, 150 mM NaCl, pH 7.4) to achieve desired LNP concentration, namely 0.005 and 0.003 mg / mL for target doses at 0.03 and 0.05 mg / mL respectively. Exemplary preparation conditions are summarized in Table 4. Exemplary liver potency of compositional variants is summarized in Table 5.

[0823]

[0330] FIG. 2A is a cryoelectron micrograph showing morphology of TA7 (47.5 / 10 / 40 / 2.5; % Molar Ratio of IL7 / Cholesterol / DSPC / DMG-PEG2000 at pH 6) of IL7 compositional variant LNPs. FIG. 2B is a cryoelectron micrograph showing morphology of TA17 (47.5 / 10 / 40 / 2.5; % Molar Ratio of IL7 / Cholesterol / DSPC / DMG-PEG2000 at pH 4) of IL 7 compositional variant LNPs, which is larger, less homogenous and shows more blebbing relative to TA7.

[0824]

[0331] This example suggests compositional variants with 55% IL7 ionizable lipid provide increased potency in liver tissue. To add, as summarized in the results, a two-streamPATENT

[0825] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0826] process wherein the mixing pH is at about pH 5.9-6.2 was found to yield smaller, stabilized LNPs with a high encapsulation efficiency relative to control LNPs prepared by a two-stream process wherein the mixing pH was less than pH 5.9.

[0827] Table 4. Preparation Conditions for IL7 Compositional Variants and Properties

[0828] TA pH of RNA Citrate % Molar Ratio Size Size Size Encapsulation aqueous concentra concentration of (nm) Growth, Growth, (%) RNA tion of of Aqueous IL7 / Cholesterol / 3x freeze2wk - mixture aqueous Phase DSPC / DMG- thaw 20C

[0829] mixture (mM) PEG2000 (nm) (nm)

[0830] (mg / mL)

[0831] 1 6 0.2 50 40 / 50 / 7.5 / 2.5 52.1 22.55 14.2 99.76 2 6 0.2 50 47.5 / 43 / 7.5 / 2 58.55 11.8 7.1 99.47 3 6 0.2 50 47.5 / 42 / 7.5 / 3 53.9 0.5 0.5 98.85 4 6 0.2 50 55 / 35 / 7.5 / 2.5 67.95 -0.65 -0.7 94.81 5 6 0.2 50 40 / 48 / 10 / 2 62.75 19.85 21.6 99.72 6 6 0.2 50 40 / 47 / 10 / 3 53.85 12.95 13.9 99.51 7 6 0.2 50 47.5 / 40 / 10 / 2.5 57.3 1.8 3.6 99.08 8 6 0.2 50 55 / 33 / 10 / 2 69.5 0.55 -0.3 96.43 9 6 0.2 50 55 / 32 / 10 / 3 65.65 -0.45 -0.4 89.75 10 6 0.2 50 40 / 45 / 12.5 / 2.5 58.1 17.65 22.2 99.54 11 6 0.2 50 47.5 / 38 / 12.5 / 2 61.2 9.1 9.35 99.34 12 6 0.2 50 47.5 / 37 / 12.5 / 3 56.25 0.1 -0.1 97.88 13 6 0.2 50 55 / 30 / 12.5 / 2.5 66.75 0.8 -0.3 91.87 14 4 0.2 50 47.5 / 42 / 7.5 / 3 67.02 -1.41 n / a 97.51 15 4 0.2 25 47.5 / 42 / 7.5 / 3 64.7 -3.2 n / a 97.02 16 4 0.1 50 47.5 / 42 / 7.5 / 3 63.99 -0.17 n / a 97.88 17 4 0.2 50 47.5 / 40 / 10 / 2.5 74.12 1.09 n / a 97.46 18 4 0.2 50 47.5 / 39.5 / 10 / 3 68.31 -0.75 n / a 97.65

[0832]

[0833] 19 4 0.2 50 47.5 / 37 / 12.5 / 3 64.47 4.15 n / a 96.2 Table 5. Liver potency of IL7 Compositional Variants four days post injection.

[0834] TA Max A to G Edit Max A to G Edit

[0835] ((%), 0.03mpk) ((%), 0.05mpk)

[0836] 1 26.2 26.5

[0837] 2 45.1 26.9

[0838] 3 20.9 25.4

[0839] 4 42.4 34.3

[0840] 5 33.3 28.0

[0841] 6 25.7 14.7

[0842] 7 43.7 28.5

[0843] 8 41.0 35.8

[0844] 9 30.5 18.7

[0845] 10 38.2 29.7

[0846] 11 40.3 14.5

[0847] 12 43.4 22.8

[0848] 13 22.4 2.6

[0849] 14 31.8 24.8

[0850] 15 31.4 30.0

[0851] 16 n / a n / a

[0852] 17 47.4 n / a

[0853] 18 38.1 22.2

[0854]

[0855] 19 29.2 18.9PATENT

[0856] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0857] Example 2. Evaluating stability of lipid nanoparticles at cold and cryogenic storage

[0332] This example illustrates size, encapsulation efficiency and stability of two compositional and process variations of IL7. LNP 1 and LNP 2 were produced by the general two-stream LNP preparation process described above. Briefly, a first solution comprising one or more nucleic acids was mixed with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid to yield a mixed product solution at pH 6.4. The lipid molar ratios of these LNPs are summarized in Table 6.

[0858] Table 6. Standard and High Ionizable Lipid (HI) Compositional Variants

[0859] LNP Composition % Molar Ratio (IL7 / Cholesterol / DSPC / DMG-PEG2000)

[0860] LN P I 47.5 / 40 / 10 / 2.5

[0861] LNP2 55 / 35 / 7.5 / 2.5

[0862]

[0863] LNP preparations were stored at -80 °C or -20 °C and evaluated at 6 and 12 months. For stability evaluation, frozen LNP stock was first allowed to completely thaw at room temperature. The particle size was then measured using dynamic light scattering (DLS). The particle encapsulation was measured using the Ribogreen assay. Results of the stability studies are summarized in Table 7 and Table 8.

[0864] Table 7. Stability Analytics at -80°C

[0865] LN P I LNP2

[0866] Particle

[0867] Particle Encapsulation Encap sulatio PDI Size PDI

[0868] Size (nm) (%) n (%) (nm)

[0869] TO 63.14 0.056 98.0 73.21 0.038 94.0 6 months 62.2 0.052 97.9 73.3 0.050 92.9

[0870]

[0871] 12 months 63.9 0.036 — 72.9 0.045 —PATENT

[0872] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0873] Table 8. Stability Analytics at -20°C.

[0874] LN P I LNP2

[0875] Particle

[0876] Particle Encapsulation Encap sulatio PDI Size PDI

[0877] Size (nm) (%) n (%) (nm)

[0878] TO 63.14 0.056 98.0 73.21 0.038 94.0 6 months 71.7 0.132 97.7 73.7 0.040 92.9

[0879]

[0880] 12 months 77.8 0.156 — 73.1 0.76 —

[0881]

[0333] This example suggests compositional variants with 55% IL7 are highly stable with respect to LNP size.

[0882] Example 3. Stability of LNPs produced by the two-stream process comprising IL76

[0883]

[0334] This example illustrates size, encapsulation efficiency and stability of two compositional and process variations of IL76. LNP 1 and LNP 2 were produced at a range of mixing pH by a two-stream LNP preparation process. Briefly, a first solution comprising one or more nucleic acids was mixed with a second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid to yield a mixed product solution at pH 4 to pH 6.4. Two exemplary total flow rates were utilized during mixing, namely, 40 mL / min. and 60 mL / min.

[0884]

[0335] Both LNP 1 and LNP 2 comprise ionizable lipid IL76, DSPC, cholesterol, and DMG-PEG and encapsulate a chemically modified sgRNA and mRNA encoding an adenine base editor. LNP 1 and LNP 2 differ in the amount of PEG lipid (2.1% vs. 2.5%) and corresponding amount of cholesterol. For both LNP 1 and LNP 2, the ratio of sgRNA to mRNA was 1:1, and the N / P ratio of formulated LNPs was 6:1. The components of LNP 1 and LNP 2 are indicated in Table 9, below.

[0885] Table 9. Components of LNP 1 and LNP 2

[0886] LNP 1 LNP 2 Component Name

[0887] (Mol %) (Mol %) Ionizable lipid IL76 in Tables 1-3 47.5 47.5

[0888] 1,2-di stearoyl-sn-gly cero-3 -phosphocholine 10 10 Phospholipid

[0889] (DSPC)

[0890] 40 40.4 Sterol Cholesterol

[0891] 1,2-dimyristoyl-rac-gly cero-3 - 2.5 2.1 PEG lipid methoxypolyethylene glycol-2000 (PEG2000-

[0892]

[0893] DMG or DMG-PEG)PATENT

[0894] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0895]

[0336] The preparation conditions and properties for LNP1 and LNP2 are indicated in Table 9 and Table 10, below as well as the particle size, encapsulation efficiency, and size growth data for the various test articles.

[0896] Table 10. Preparation Conditions for LNP 1 Test Articles and Properties

[0897] Test LNP pH of Flow rate Size Size Encapsulation Article aqueous lipid ethanol (nm) growth, 3x efficiency RNA mixture freeze(%) mixture thaw (nm)

[0898] 1-1 LNP 1 4 10 ml / min 79.66 14.54 91.5 1-2 LNP 1 5.7 10 ml / min 67.75 23.03 90.9 1-3 LNP 1 5.7 15 ml / min 66.8 13.71 88.2 1-4 LNP 1 6.0 10 ml / min 59.34 12.17 97.5 1-5 LNP 1 6.0 15 ml / min 58.63 7.58 96.9 1-6 LNP 1 6.3 10 ml / min 96.13 3.55 67.3

[0899]

[0900] 1-7 LNP 1 6.3 15 ml / min 124.6 -3.7 45.6

[0901] Table 11. Preparation Conditions for LNP 2 Test Articles and Properties

[0902] Test LNP pH of Flow rate Size (nm) Size Encapsulation Article aqueous lipid ethanol growth, 3x efficiency RNA mixture freeze(%) mixture thaw (nm)

[0903] 2-1 LNP 2 4 10 ml / min 80.27 27.73 92.7 2-2 LNP 2 5.8 10 ml / min 64.34 26.56 92.9 2-3 LNP 2 5.8 15 ml / min 62.95 24.55 94 2-4 LNP 2 6.1 10 ml / min 63.49 4.23 97.8 2-5 LNP 2 6.1 15 ml / min 66.7 3.73 97.9 2-6 LNP 2 6.3 10 ml / min 136.1 - 30.3

[0904]

[0905] 2-7 LNP 2 6.3 15 ml / min 172.4 - 24.5

[0906]

[0337] The data in Tables 10 and 11 is depicted in FIG. 3A-FIG. 3C. FIG. 3A is a graph comparing size of LNPs relative to mixing pH. FIG. 3B is a graph comparing encapsulation efficiency of LNPs relative to mixing pH. FIG. 3C is a graph comparing stability as measured by change in size of LNP particles after three cycles of freeze and thaw.

[0907]

[0338] Mixing at a pH near 6.0-6.1 produced small (for example, about 60-70 nm), high encapsulation (for example, greater than 90% or greater than 95%), stable LNPs (for example minimal change in size after three rounds of freeze and thaw). Lower mixing pH reduced encapsulation and colloidal stability as shown in FIG. 3B and FIG. 3C. Mixing at higher pH, for example, greater than 6.1, resulted in poor LNP formation. No difference inPATENT

[0908] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0909] any parameters tested was seen between compositions comprising 2.1% PEG and 2.5% PEG or flow rate of 10 mL / min. and 15 mL / min.

[0910]

[0339] The results in FIG. 4A-FIG. 4D show heat maps of LNP compositions comprising exemplary ionizable lipid IL76 and PEG concentration ranges when mixed at pH 6 relative to control mixing at pH 4. The LNP formulations encapsulate chemically modified sgRNA and mRNA encoding an adenine base editor. For all LNP formulations, the ratio of sgRNA to mRNA was 1:1, and the N / P ratio was 6. LNP formulations differed in the amount of PEG lipid (1.9%, 2.1%, 2.5%) and ionizable lipid (47.5%, 51.25%, 55%).

[0911]

[0340] FIG. 4A is a heat map comparing size of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG and IL76 between 47.5-55% at mixing pH 6 relative to a control mixing pH 4. FIG. 4B is a heat map comparing encapsulation efficiency of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG and IL76 between 47.5-55% at mixing pH 6 relative to a control mixing pH 4. The data used to create the heat maps in FIGS. 4A and 4B is provided in Table 12. Table 12 also provides PDI data for the various LNP formulations.

[0912]

[0341] FIG. 4C is a heat map comparing size growth of fresh LNPs subjected to three rounds of freeze-thaw of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG and IL76 between 47.5-55% at mixing pH 6 relative to a control mixing pH 4. The heat map in FIG. 4C was generated by using the particle size data in Table 12 as time zero relative to the 3x freeze thaw particle size data in Table 13. Table 13 also contains additional particle size data and encapsulation efficiency data for the LNP formulations.

[0913]

[0342] FIG. 4D is a heat map comparing size growth after storage for two weeks at -20 °C, and then subjected to three rounds of freeze-thaw of IL76 LNPs comprising between 1.9-2.5% exemplary DMG-PEG and IL76 between 47.5-55% at mixing pH 6 relative to a control mixing pH 4. The heat map in FIG. 4D was generated by using the particle size data in Table 12 as time zero relative to the -20°C particle size data in Table 14. Table 14 also contains PDI and encapsulation efficiency data for the LNP formulations after 2 weeks.

[0914]

[0343] The results depicted in the heat maps in FIGS. 4A and 4B showed that LNPs comprising 47.5%-51.25% IL76 ionizable lipid mixed at pH 6 were smaller and have higher encapsulation efficiency than those mixed at pH 4. The results depicted in heat maps in FIGS. 4C and 4D showed that the LNPs had similar stability across all test formulations and mixing pHs.PATENT

[0915] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0916] Table 12. Pre-freeze LNP Analytics

[0917] Formulation Composition Particle PDI Encapsulation

[0918] Ratio (IL / PEG Size (%)

[0919] Lipid / DSPC / Cho (nm)

[0920] lesterol

[0921] LN P I 47.5 / 1.9 / 10 / 40.6 66.38 0.069 95.8

[0922] LNP2 47.5 / 2.2 / 10 / 40.3 63.61 0.079 95.5

[0923] LNP3 47.5 / 2.5 / 10 / 40 63.46 0.074 94.9

[0924] LNP4 51.25 / 1.9 / 10 / 36. 66.42 0.047 95.7

[0925] 85

[0926] LNP5 51.25 / 2.2 / 10 / 36. 63.85 0.063 94.5

[0927] 55

[0928] LNP6 51.25 / 2.5 / 10 / 36. 63.1 0.049 92.8

[0929] 25

[0930] LNP7 55 / 1.9 / 10 / 33.1 73.83 0.026 89.7

[0931] LNP8 55 / 2.2 / 10 / 32.8 67.81 0.038 90.3

[0932] LNP9 55 / 2.5 / 10 / 32.5 68.79 0.042 84.5

[0933]

[0934] LNP 10* 47.5 / 2.5 / 10 / 40 81.7 0.072 86.6

[0935] * = pH 4 LNP formulation

[0936] Table 13. Freeze Thaw Analytics at -80°C

[0937] 3X Freeze-Thaw Cycles 4 weeks 12 weeks Formulation Particle PDI Encapsulation Particle PDI Encapsulation Particle PDI Encapsulation Size (%) Size (%) Size (%) (nm) (nm) (nm)

[0938] LNP1 70.23 0.065 95.8 67.35 0.031 96.6 — — LNP2 65.06 0.047 95.0 65.97 0.067 96.1 — — — LNP3 64.06 0.063 94.8 64.93 0.084 95.5 64.44 0.072 95.6 LNP4 66.43 0.057 95.5 70.14 0.048 96.3 — — — LNP5 63.99 0.056 94.3 65.95 0.054 94.9 — — — LNP6 63.03 0.043 92.3 65.52 0.052 93.5 — — — LNP7 71.52 0.051 88.9 75.51 0.06 90.3 — — — LNP8 67.39 0.041 88.5 71.68 0.029 90.0 — — — LNP9 67.9 0.038 83.8 70.82 0.06 85.3 — — — LNP10* 82.35 0.084 82.9 81.76 0.084 85.8 81.73 0.083 85.1

[0939]

[0940] * = pH 4 LNP formulationPATENT

[0941] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0942] Table 14. Freeze Thaw Analytics at -20°C

[0943] Formulation 2 weeks

[0944] Particle Size

[0945] PDI Encapsulation (%)

[0946] (nm)

[0947] LN P I 71.09 0.081 96.6

[0948] LNP2 66.42 0.085 95.9

[0949] LNP3 63.92 0.083 95.6

[0950] LNP4 67.91 0.06 96.3

[0951] LNP5 64.49 0.038 95.1

[0952] LNP6 65.28 0.039 93.5

[0953] LNP7 76.88 0.039 90.2

[0954] LNP8 71.56 0.031 89.9

[0955] LNP9 73.67 0.077 86.0

[0956]

[0957] LNP 10* 82.06 0.077 86.8

[0958] * = pH 4 LNP formulation

[0959]

[0344] The morphology of prototype and stabilized IL76 LNPs was tested and results are depicted in cryoelectron micrographs in FIG. 5A-FIG. 5B.

[0960]

[0345] FIG. 5A shows a cryoelectron micrograph showing morphology of prototype IL76 LNPs comprised of 2.5% DMG-PEG and formulated with mixing pH 4. FIG. 5B shows a cryoelectron micrograph showing morphology of stabilized IL76 LNPs comprised of 2.5% DMG-PEG and formulated with mixing pH6, which is smaller, more homogenous and shows less blebbing relative to prototype IL76 LNPs.

[0961] Example 4. Stability of LNPs produced by the two-stream process comprising IL477

[0962]

[0346] By the same approach with the two-stream mixing process at 40 mL / min total flow rate, another exemplary IL477 ionizable lipid, showed similar improvements. FIG. 6A-FIG. 6B shows heat maps of compositions comprising exemplary ionizable lipid and PEG concentration ranges when mixed at pH 6 relative to control mixing at pH 4. FIG. 6A is a heat map comparing size growth of fresh LNPs subjected to three rounds of freeze-thaw IL477 LNPs comprising between 2-3% exemplary DMG-PEG and 47.5-55% IL477 at mixing pH 6 relative to a control mixing pH 4. The data used to create the heat map in FIG. 6A is provided in Table 15. Table 15 also provides PDI data for the various LNP formulations. Table 16 provides particle size data after 3x freeze thaw cycles, after 4 weeks, and after 12 weeks for the various LNP formulations.PATENT

[0963] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0964]

[0347] FIG. 6B is a heat map comparing size growth after storage for two weeks at - 20 °C, and then subjected to three rounds of freeze-thaw of IL477 LNPs comprising between 2-3% exemplary DMG-PEG at mixing pH 6 relative to a control mixing pH 4. The heat map in FIG. 6B was generated by using the particle size data in Table 15 as time zero relative to the 3x freeze thaw -20°C particle size data in Table 16. Table 16 also contains additional PDI and encapsulation efficiency data for the LNP formulations after 2 weeks.

[0965] Table 15. Pre-freeze LNP Analytics

[0966] Composition Ratio

[0967] Particle Size Encapsulation Formulation (IL / PEG PDI

[0968] (nm)

[0969] Lipid / DSPC / Cholesterol (%) LN P I 47.5 / 2.0 / 10 / 40.5 70.57 0.059 97.8 LNP2 47.5 / 2.5 / 10 / 40 65.88 0.041 97.8 LNP3 47.5 / 3 / 10 / 39.5 61.59 0.057 97.6 LNP4 51.25 / 2 / 10 / 36.75 65.92 0.044 97.9 LNP5 51.25 / 2.5 / 10 / 36.25 62.96 0.06 97.8 LNP6 51.25 / 3 / 10 / 35.75 60.31 0.045 97.7 LNP7 55 / 2.0 / 10 / 33 65.94 0.036 97.8 LNP8 55 / 2.5 / 10 / 32.5 66.07 0.028 97.7 LNP9 55 / 3 / 10 / 32 66.18 0.064 97.0

[0970]

[0971] LNP 10* 47.5 / 2.5 / 10 / 40 79.99 0.025 98.1 * = pH 4 LNP formulationPATENT

[0972] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0973] Table 16. Freeze Thaw Analytics at -80°C

[0974] 3X Freeze-Thaw Cycles 4 weeks 12 weeks Formulation Particle Particle Particle

[0975] Encapsulation Encapsulation Encapsulation Size PDI Size PDI Size PDI (nm) (%) (nm) (%) (nm) (%) LNP1 97.64 0.035 97.1 86.01 0.04 97.6 — — — LNP2 84.25 0.089 97.6 72.89 0.052 97.8 — — — LNP3 68.01 0.07 97.5 64.17 0.069 97.7 — — — LNP4 79.62 0.077 97.8 71.92 0.055 97.8 — — — LNP5 67.56 0.077 97.7 63.24 0.047 97.9 — — — LNP6 62.45 0.071 97.7 61.19 0.052 98.0 — — — LNP7 74.79 0.064 97.6 71.14 0.029 97.8 — — — LNP8 66.52 0.056 98.1 66.98 0.036 98.0 67.8 0.039 98.0 LNP9 64.69 0.062 97.2 64.74 0.049 97.5 — — —

[0976]

[0977] LNP10* 91.51 0.054 97.5 83.68 0.039 98.2 84.88 0.030 97.8 * = pH 4 LNP formulation

[0978] Table 17. Freeze Thaw Analytics at -20°C

[0979] 2 weeks

[0980] Formulation Particle Size Encapsulation

[0981] PDI

[0982] (nm) (%)

[0983] LN P I 107.2 0.042 97.9

[0984] LNP2 92.7 0.043 98.2

[0985] LNP3 81.56 0.097 98.0

[0986] LNP4 95.31 0.053 98.2

[0987] LNP5 72.39 0.092 97.9

[0988] LNP6 65.33 0.053 97.9

[0989] LNP7 86.09 0.083 97.8

[0990] LNP8 69.47 0.031 97.9

[0991] LNP9 64.58 0.033 97.3

[0992]

[0993] LNP 10* 102.9 0.057 98.5

[0994] * = pH 4 LNP formulation

[0995]

[0348] The morphology of stabilized IL477 LNPs shown in FIG. 7B shows smaller more homogenous LNPs with less blebbing relative to prototype IL477 LNPs shown in FIG. 7A.

[0996]

[0349] FIG. 7A shows a cryoelectron micrograph showing morphology of prototype IL477 LNPs comprised of 47.5% IL477 ionizable lipid, 2.5% DMG-PEG and formulated with mixing pH 4. FIG.7B shows a cryoelectron micrograph showing morphology of stabilized IL477 LNPs, compromised of 55% IL477 ionizable lipid, 2.5% DMG-PEG andPATENT

[0997] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[0998] formulated with mixing pH 6, which is smaller, more homogenous and shows less blebbing relative to prototype IL477 LNPs.

[0999]

[0350] These results show that a two-stream process wherein the mixing pH of about pH 5.9-6.2 yielded smaller, stabilized LNPs with a high encapsulation efficiency relative to control LNPs prepared by a two-stream process wherein the mixing pH was less than pH 5.9.

[1000] Example 5. Stability of LNPs produced by the two-stream process after storage at -80 °C and -20 °C for three months

[1001]

[0351] This example illustrates stability after freeze-thawing of LNPs produced by the improved two-stream process described in Examples 1. Briefly, LNPs were tested after three rounds of freeze and thaw after storage at -80 °C and -20 °C for three months.

[1002]

[0352] Exemplary IL76 and IL477 LNPs were tested, and the composition of the components is shown in Table 18. As shown in Tables 19 and 20, below, there was no appreciable change in size after freeze-thaw in the LNPs stored at -80 °C for three months. LNPs stored at -20 °C for one month showed a slight increase in size, which was maintained up to storage for 3 months.

[1003] Table 18. Tested Composition Ratios

[1004] Test Article Name TA 1 TA 2

[1005] Ionizable Lipid IL76 IL477

[1006] Composition Ratio 47.5 / 10 / 40 / 2.5 55 / 10 / 32.5 / 2.5 (IZ / DSPC / Cholesterol / PEG

[1007] Lipid)

[1008]

[1009] RNA mix 50mM Citrate; pH 6 50mM Citrate; pH 6

[1010] Table 19. Freeze Thaw Analytics at -80°C

[1011] TA 1 TA 2

[1012] IL76; pH6 IL477; pH 6

[1013] Avg. Avg. Editin Editin g in g in Partici Encap sulatio Partici Encap sulatio Mice Mice e Size PDI n e Size PDI n

[1014] at 0.1 at 0.1 (nm) (%) (nm)

[1015] mg / kg (%) mg / kg dose dose (%) (%) 0.06 0.05

[1016] TO 67.84 95.4 41.5 73.92 95.6 31.8

[1017]

[1018] 3 4PATENT

[1019] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[1020] TA 1 TA 2

[1021] IL76; pH6 IL477; pH 6

[1022] Avg. Avg. Editin Editin g in g in Partici Encap sulatio Partici Encap sulatio Mice Mice e Size PDI n e Size PDI n

[1023] at 0.1 at 0.1 (nm) (%) (nm)

[1024] mg / kg (%) mg / kg dose dose (%) (%) 3

[1025] 0.06 0.06

[1026] month 67.16 96.5 35.7 74.79 97.3 21.2

[1027] 8 6

[1028] s

[1029] 6

[1030] 0.10 0.06

[1031] month 77.69 95.0 21.5 68.81 97.0 17.3

[1032] 5 5

[1033]

[1034] s

[1035] Table 20. Freeze Thaw Analytics at -20°C

[1036] TA 1 TA 2

[1037] IL76 SD; pH6 L477 SD; pH 6

[1038] Avg. Avg.

[1039] Editin Editin

[1040] g in g in

[1041] Partici Partici Mice Encap sulatio Mice Encap sulatio e Size PDI e Size PDI at 0.1 at 0.1 n (%)

[1042] (nm) n (%) (nm) mg / kg mg / kg

[1043] dose dose

[1044] (%) (%)

[1045] 0.06 0.05

[1046] TO 67.84 95.4 41.5 73.92 95.6 31.8

[1047] 3 4

[1048] 3

[1049] 0.07 0.10

[1050] month 70.22 96.4 32.8 79.52 97.1 20.4

[1051] 6 0

[1052] s

[1053] 6

[1054] 0.08

[1055] month — — — — 69.03 97.0 12.8

[1056] 5

[1057]

[1058] s

[1059]

[0353] Overall, results showed that the LNPs produced by the improved two-stream mixing process were stable after storage at both -20 °C and -80 °C for at least 3 months.

[1060]

[0354] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed toPATENT

[1061] ATTORNEY DOCKET NO.: BLP-1034-10-WO

[1062] be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

Claims

PATENTATTORNEY DOCKET NO.: BLP-1034-10-WOCLAIMSWhat is claimed is:

1. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP.

2. The LNP of claim 1, wherein the ionizable lipid comprises from about 54 mol % to about 57 mol % of the total lipid present in the LNP.

3. The LNP of claim 1, wherein the phospholipid comprises from about 7 mol% to about 8 mol% of the total lipid present in the LNP.

4. The LNP of claim 1, wherein the cholesterol lipid or derivative thereof comprises from about 35 mol % to about 40 mol % of the total lipid present in the LNP.

5. The LNP of claim 1, wherein the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

6. The LNP of any one of the preceding claims, wherein the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO7. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP,wherein the LNP is prepared by mixing the nucleic acid cargo, the ionizable lipid, the phospholipid, the cholesterol lipid or derivative thereof, and the PEG lipid at a pH of between 5.9 and 6.2.

8. The LNP of claim 7, wherein the ionizable lipid comprises about 47.5 mol % of the total lipid present in the particle9. The LNP of claim 7, wherein the phospholipid comprises from about 9.5 mol% to about 10 mol% of the total lipid present in the particle.

10. The LNP of claim 7, wherein the cholesterol lipid or derivative thereof comprises from about 35 mol % to about 40 mol % of the total lipid present in the particle.

11. The LNP of claim 7, wherein the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the particle.

12. The LNP of any one of claims 7-11, wherein the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO13. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the particle.wherein the hydrodynamic diameter of the LNP increases by less than about 10 nm after at least one freeze thaw cycle.

14. The LNP of claim 13, wherein the hydrodynamic diameter of the LNP increases by less than about 10 nm after two freeze thaw cycles.

15. The LNP of claim 13, wherein the hydrodynamic diameter of the LNP increases by less than about 10 nm after three freeze thaw cycles.

16. The LNP of any one of claims 13-15, wherein the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

17. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP.wherein the hydrodynamic diameter of the LNP increases by less than about 20% after at least one freeze thaw cycle.

18. The LNP of claim 17, wherein the hydrodynamic diameter of the LNP increases by less than about 20% after two freeze thaw cycles.

19. The LNP of claim 17, wherein the hydrodynamic diameter of the LNP increases by less than about 20% after three freeze thaw cycles.

20. The LNP of claim 17, wherein the hydrodynamic diameter of the LNP increases by less than about 15% after three freeze thaw cycles.

21. The LNP of claim 17, wherein the hydrodynamic diameter of the LNP increases by less than about 10% after three freeze thaw cycles.

22. The LNP of claim 17, wherein the hydrodynamic diameter of the LNP increases by less than about 5% after three freeze thaw cycles.

23. The LNP of any one of claims 7-22, wherein the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the particle; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

24. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 51 mol % to about 60 mol % of the total lipid present in the LNP;PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP.wherein the hydrodynamic diameter of the LNP increases by less than about 20% after storage at -20°C for up to 4 weeks, orwherein the hydrodynamic diameter of the LNP increases by less than about 10% after storage at -80°C for up to 3 months.

25. The LNP of claim 24, wherein the hydrodynamic diameter of the LNP increases by less than about 15%, about 10%, about 5%, or about 1% after storage at -20°C for up to 4 weeks.

26. The LNP of claim 24, wherein the hydrodynamic diameter of the LNP increases by less than about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% after storage at -80°C for up to 3 months.

27. The LNP of any one of claims 24-26, wherein the ionizable lipid comprises about 55 mol % of the total lipid present in the LNP; the phospholipid comprising from about 7.5 mol % to about 8 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 35 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2.5 mol % to about 3 mol % of the total lipid present in the LNP.

28. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP,wherein the hydrodynamic diameter of the LNP increases by less than about 15 nm after at least one freeze thaw cycle.

29. The LNP of claim 28, wherein the hydrodynamic diameter of the LNP increases by less than about 15 nm after two freeze thaw cycles.

30. The LNP of claim 28, wherein the hydrodynamic diameter of the LNP increases by less than about 15 nm after three freeze thaw cycles.

31. The LNP of any one of claims 28-30, wherein the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

32. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP,wherein the hydrodynamic diameter of the LNP increases by less than about 20% after at least one freeze thaw cycle.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO33. The LNP of claim 32, wherein the hydrodynamic diameter of the LNP increases by less than about 20% after two freeze thaw cycles.

34. The LNP of claim 32, wherein the hydrodynamic diameter of the LNP increases by less than about 20% after three freeze thaw cycles.

35. The LNP of claim 32, wherein the hydrodynamic diameter of the LNP increases by less than about 15% after three freeze thaw cycles.

36. The LNP of claim 32, wherein the hydrodynamic diameter of the LNP increases by less than about 10% after three freeze thaw cycles.

37. The LNP of claim 32, wherein the hydrodynamic diameter of the LNP increases by less than about 5% after three freeze thaw cycles.

38. The LNP of any one of claims 32-37, wherein the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

39. A lipid nanoparticle (LNP) comprising:(a) a nucleic acid cargo;(b) an ionizable lipid comprising from about 47 mol % to about 60 mol % of the total lipid present in the LNP;(c) a phospholipid comprising from about 7 mol % to about 10 mol % of the total lipid present in the LNP;(d) a cholesterol lipid, or derivative thereof, comprising from about 30 mol % to about 40 mol % of the total lipid present in the LNP; and(e) a PEG lipid comprising from about 1 mol % to about 5 mol % of the total lipid present in the LNP,PATENTATTORNEY DOCKET NO.: BLP-1034-10-WOwherein the hydrodynamic diameter of the LNP increases by less than about 20% after storage at -20°C for up to 4 weeks, orwherein the hydrodynamic diameter of the LNP increases by less than about 10% after storage at -80°C for up to 3 months.

40. The LNP of claim 39, wherein the hydrodynamic diameter of the LNP increases by less than about 15%, about 10%, about 5%, or about 1% after storage at -20°C for up to 4 weeks.

41. The LNP of claim 39, wherein the hydrodynamic diameter of the LNP increases by less than about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1% after storage at -80°C for up to 3 months.

42. The LNP of any one of claims 39-41, wherein the ionizable lipid comprises about 47.5 mol % of the total lipid present in the LNP; the phospholipid comprising from about 9.5 mol % to about 10 mol% of the total lipid present in the LNP; the cholesterol lipid, or derivative thereof, comprises about 40 mol % of the total lipid present in the LNP; and the PEG lipid comprises from about 2 mol % to about 3 mol % of the total lipid present in the LNP.

43. The LNP of any one of claims 13-42, wherein the encapsulation efficiency after storage is greater than about 90%.

44. The LNP of claim 43, wherein the encapsulation efficiency after storage is greater than 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

45. The LNP of any one of the preceding claims, wherein the ionizable lipid is selected from Table 1.

46. The LNP of any one of the preceding claims, wherein the ionizable lipid is selected from Table 2.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO47. The LNP of any one of the preceding claims, wherein the phospholipid comprises 1,2- dioleoyl-sw-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl PE), 1,2- distearoyl-sw-glycero-3-phosphocholine (DSPC), cholesterol, 1,2-distearoyl-sn- glycero-3 -phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (succinyl -DPPE), l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC), or a combination thereof.

48. The LNP of claims 47, wherein the phospholipid comprises distearoylphosphatidylcholine (DSPC).

49. The LNP of any one of the preceding claims, wherein the PEG lipid comprises PEG- c-DMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEG- DSPE, or any combination thereof.

50. The LNP of claims 49, wherein the PEG lipid comprises a PEG-dimyristylglycerol (PEG-DMG) conjugate.

51. The LNP of any one of the preceding claims, wherein the nucleic acid cargo comprises between about 3000 to about 8000 nucleotides (nts).

52. The LNP of claim 51, wherein the nucleic acid cargo comprises between about 5000 to about 7000 nucleotides.

53. The LNP of any one of the preceding claims, wherein the nucleic acid cargo comprises a guide RNA and an mRNA encoding a CRISPR base editor.

54. The LNP any one of the preceding claims, wherein the N: P ratio of between about 10:1 to about 5:1.

55. The LNP of any one of the preceding claims, wherein the LNP has an average hydrodynamic diameter between about 40 nm to about 100 nm.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO56. The LNP of claim 14, wherein the LNP has an average hydrodynamic diameter between about 50 nm to about 70 nm.

57. The LNP of any one of the preceding claims, wherein the polydispersity index (PDI) is less than about 0.1.

58. The LNP of claim 57, wherein the polydispersity index (PDI) is less than about 0.05.

59. A method of preparing a lipid nanoparticle (LNP) comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, wherein:the first solution comprises one or more nucleic acids, andthe second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid;wherein the mixing step is carried out to yield a mixed solution at a pH of between about 5.9 to 6.2,thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

60. A method of preparing a lipid nanoparticle (LNP) comprising a nucleic acid cargo, the method comprising a mixing step combining a first solution and a second solution, whereinthe first solution comprises one or more nucleic acids,the second solution comprising an ionizable lipid, a phospholipid, a cholesterol lipid, and a PEG lipid;wherein the mixing step is carried out to yield a mixed solution at a pH of within 1 unit of the pKa of the ionizable lipid,thereby providing a product comprising a lipid nanoparticle encapsulating a nucleic acid cargo.

61. The method of claim 59 or 60, wherein the pH of the first solution is between pH 5.5 to pH 6.5 prior to the mixing step.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO62. The method of claims 59-61, wherein the method further comprises a step of adjusting the pH of the first solution and / or the second solution to within 0.5 units of each other prior to the mixing step.

63. The method of any one of claims 59-62, wherein the LNP comprises an N: P ratio of between about 10:1 to about 5:1.

64. The method of any one of claims 59-63, wherein the LNP has a hydrodynamic diameter between of about 40 nm to about 100 nm.

65. The method of claim 64, wherein the LNP has a hydrodynamic diameter of about 50 nm to 70 nm.

66. The method of any one of claims 59-65, wherein the encapsulation efficiency of the LNP is at least about 90%.

67. The method of any one of claims 59-66, wherein the polydispersity index (PDI) of the LNP is less than about 0.1.

68. The method of any one of claims 59-67, wherein the first solution is an aqueous solution of water: ethanol in about 3: 1 (v / v) ratio, about 4: 1 (v / v) ratio, or about 2: 1 (v / v) ratio.

69. The method of any one of claim 59-68, wherein the second solution comprises:(a) about 51 mol % to about 55 mol % of an ionizable lipid;(b) about 7 mol % to about 10 mol % of a phospholipid;(c) about 30 mol % to about 40 mol % of a cholesterol lipid, or derivative thereof; and (d) about 2 mol % to about 3 mol % of a PEG lipid.

70. The method of claim 69, wherein the second solution comprises about 55 mol % of the ionizable lipid; about 7.5 mol % to about 8 mol% of the phospholipid; about 35% of the cholesterol lipid, or derivative thereof; and about 2.5 mol % to about 3 mol % the PEG lipid.PATENTATTORNEY DOCKET NO.: BLP-1034-10-WO71. The method of any one of claim 59-70, wherein the second solution comprises:(a) about 47 mol % to about 60 mol % of an ionizable lipid;(b) about 7 mol % to about 10 mol % of a phospholipid;(c) about 30 mol % to about 40 mol % of a cholesterol lipid, or derivative thereof; and (d) about 2 mol % to about 3 mol % of a PEG lipid.

72. The method of claim 71, wherein the second solution comprises about 47.5 mol % of the ionizable lipid; about 9.5 mol % to about 10 mol% of the phospholipid; about 40% of the cholesterol lipid, or derivative thereof; and about 2.5 mol % to about 3 mol % the PEG lipid.

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