Lipid nanoparticles for intracellular delivery of proteins

Abstract

Lipid nanoparticle formulations for delivery of therapeutic agents can be used in the treatment of diseases or disorders.

Description

DOCKET NO.: 348358.18102 LIPID NANOPARTICLES FOR INTRACELLULAR DELIVERY OF PROTEINS The present application claims priority from U.S. provisional application no. 63 / 575,830 filed April 7, 2024. This application is incorporated by reference herein in its entirety. FIELD

[0001] The present disclosure relates in general to formulations of lipid nanoparticles (LNPs). The disclosure relates in particular to intracellular delivery of therapeutic agents, e.g., proteins in vivo or in vitro. BACKGROUND

[0002] Protein-based therapeutics for intracellular targets, such as the CRISPR-Cas9 system, have been employed in gene and immune therapies for diverse diseases. Despite considerable efforts, an efficient delivery system for the in vivo intracellular delivery of protein therapeutics is still lacking. SUMMARY

[0003] In one aspect, new lipid nanoparticle (LNP) formulations are provided.

[0004] In preferred aspects, the LNPs encapsulate or coordinate with one or more therapeutic agent for in vivo intracellular delivery of the therapeutic agent.

[0005] In certain aspects, a lipid nanoparticle (LNP) formulation comprises: an ionizable lipid, at least one helper lipid, a polyethylene glycol (PEG)-modified lipid, a sterol, and at least one gene editing agent. In certain embodiments, the gene editing agent is a CRISPR-Cas9 gene editing system. In certain embodiments a single guide RNA (sgRNA) targets a specific nucleic acid sequence.

[0006] In one aspect, a lipid nanoparticle (LNP) formulation comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid and a sterol.

[0007] In certain embodiments, the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

[0008] In certain embodiments, the cationic lipid comprises 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof. In certain embodiments, the zwitterionic lipid comprises 1,2-dioleoyl-sn- 170450739.1glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof. In certain embodiments, the anionic lipid comprises 1,2-dimyristoyl-sn- glycero-3-phosphate (14PA), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho (1’-rac-glycerol) (18PG) or the combination thereof.

[0009] In certain embodiments, the ionizable lipid comprises DLin-MC3-DMA.

[0010] In certain embodiments, the PEGylated lipid comprises myristoyl diglyceride (DMG-PEG) such as DMG-PEG 2000. In some embodiments, the DMG-PEG may have a molecular weight ranging from about 200 to about 2000 daltons.

[0011] In certain embodiments, the sterol comprises cholesterol.

[0012] In certain embodiments, the LNP formulation comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 10% to about 90%. In certain embodiments, the LNP formulation comprises a combined molar percentage of DLin-MC3-DMA and the at least one helper lipid in a range from about 20% to about 80%.

[0013] In certain embodiments, the LNP formulation comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300. In certain embodiments, the LNP formulation comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 10 to about 200.

[0014] In certain embodiments, the LNP formulation comprises a weight ratio of DLin- MC3-DMA to the helper lipid, from 0.1 to 200. In certain embodiments, the LNP formulation comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

[0015] In certain aspects, the steroid comprises a sterol. In particular aspects, the sterol comprises cholesterol.

[0016] In certain aspects, the ionizable cationic lipid comprises Dlin-MC3-DMA.

[0017] In certain aspects, the helper lipid is selected from a cationic lipid, a zwitterionic lipid, and an anionic lipid.

[0018] In certain aspects, the cationic lipid is selected from 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and dimethyl di octadecyl ammonium (DDAB).

[0019] In certain aspects, the zwitterionic lipid is selected from 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and1 ,2-distearoyl-sn -glycero-3-phosphocholine (DSPC). 2 170450739.1

[0020] In certain aspects, the anionic lipid comprises a phospholipid. In particular aspects, the phospholipid is selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and 1- stearoyl- 2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0021] In particular aspects, the PEGylated lipid comprises dimyristoyl glycerol (DMG)- polyethyleneglycol (PEG) 2000 (DMG-PEG2000).

[0022] In another aspect, a lipid nanoparticle further comprises a therapeutic agent. In certain embodiments, the therapeutic agent is encapsulated by the LNP. In certain embodiments, the LNP comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50. In certain embodiments, the LNP comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30. In certain embodiments, the therapeutic agent comprises carbohydrates, protein, lipids, nucleic acids or combinations thereof.

[0023] In another aspect, a method of intracellular delivery of a therapeutic agent comprises contacting a cell or administering to a subject a lipid nanoparticle comprising an effective amount of a therapeutic agent. In certain embodiments, the lipid nanoparticle (LNP) comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid, a sterol or combinations thereof. In certain embodiments, the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof. In certain embodiments, the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof. In certain embodiments, the zwitterionic lipid comprises 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof. In certain embodiments, the anionic lipid comprises 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), 1-stearoyl-2-oleoyl-sn- glycero-3-phospho (1’-rac-glycerol) (18PG) or the combination thereof. In certain embodiments, the ionizable lipid comprises DLin-MC3-DMA. In certain embodiments, the PEGylated lipid comprises DMG-PEG 2000. In certain embodiments, the sterol comprises cholesterol. In certain embodiments, the therapeutic agent is encapsulated by the LNP. In certain embodiments, the therapeutic agent comprises a carbohydrates, protein, lipids, nucleic acids or combinations thereof.

[0024] In another aspect, a method of treating a subject, comprises administering to the subject a lipid nanoparticle comprising a therapeutic agent embodied herein, and treating the subject. In certain embodiments, the therapeutic agent comprises a carbohydrates, protein, lipids, 3 170450739.1nucleic acids or combinations thereof. In certain embodiments, the therapeutic agent is a gene editing molecule. All of or a portion of the therapeutic agent nucleic acid may be encapsulated in the lipid nanoparticles. In some embodiments, the method yields a nucleic acid encapsulation efficiency of at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater).

[0025] In another aspect, a pharmaceutical composition comprises an LNP. In certain embodiments, the LNP comprises a therapeutic agent. For example, a gene editing agent.

[0026] In another aspect, the lipid nanoparticles have a mean diameter between 60 nm and 100 nm (e.g., between 62 nm and 98 nm, between 64 nm and 96 nm, between 66 nm and 94 nm, between 68 nm and 92 nm, about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, about 75 nm, about 76 nm, about 77 nm, about 78 nm, about 79 nm, about 80 nm, about 81 nm, about 82 nm, about 83 nm, about 84 nm, about 85 nm, about 86 nm, about 87 nm, about 88 nm, about 89 nm, about 90 nm, about 91 nm, about 92 nm, about 93 nm, about 94 nm, about 95 nm, about 96 nm, about 97 nm, about 98 nm, about 99 nm, or about 100 nm) and a polydispersity index of 0.25 or less (e.g., 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, or less).

[0027] In another aspect, an LNP comprises ionizable lipid ranging from about 5% to about 95%, helper lipid from about 0% to about 80%, cholesterol from about 1% to about 80%, and PEGylated lipid from about 0% to about 20% by weight. In certain embodiments, an LNP comprises ionizable lipid ranging from about 10% to about 90%, helper lipid from about 0% to about 60%, cholesterol from about 10% to about 60%, and PEGylated lipid from about 0% to about 10% by weight.

[0028] In some embodiments of the nanoparticle, the steroid has a molar ratio to the PEGylated lipid between about 10 and about 900, including a molar ratio of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 4 170450739.1640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, and 900.

[0029] In some embodiments of the nanoparticle, the ionizable cationic lipid has a molar ratio to the helper lipid between about 1 to about 200, including a molar ratio of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200.

[0030] In some embodiments, the nanoparticle comprise a total percentage of the ionizable lipid and the helper lipid is between about 20% and about 80%, including a total percentage of about 20%, 21%, 22%, 23%, 25%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 695, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80%.

[0031] In some embodiments, the solid nanoparticle comprises an N to P ratio between about 2 and about 14, including an N to P ratio between about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, and 14.

[0032] In some embodiments, the solid nanoparticle comprises a weight fraction of siRNA in the nucleic acid payload between about 0 to about 1, including about 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75. 0.8, 0.85, 0.9, 0.95, and about 1.

[0033] In some embodiments, the nanoparticle has a size smaller than about 400 nm, including a size of about 400 nm, 395 nm, 390 nm, 385 nm, 380 nm, 375 nm, 370 nm, 365 nm, 360 nm, 355 nm, 350 nm, 345 nm, 340 nm, 335 nm, 330 nm, 325 nm, 320 nm, 315 nm, 310 nm, 305 nm, 300 nm, 295 nm, 290 nm, 285 nm, 280 nm, 275 nm, 270 nm, 265 nm, 260 nm, 255 nm, 250 nm, 245 nm, 240 nm, 235 nm, 230 nm, 225 nm, 220 nm, 215 nm, 210 nm, 205 nm, 200 nm, 195 nm, 190 nm, 185 nm, 180 nm, 175 nm, 170 nm, 165 nm, 160 nm, 155 nm, 150 nm, 145 nm, 140 nm, 135 nm, 130 nm, 125 nm, 120 nm, 115 nm, 110 nm, 105 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, and 5 nm.

[0034] In certain aspects, the steroid comprises cholesterol; the ionizable cationic lipid comprises DLin-MC3-DMA; the PEGylated lipid comprises DMG-PEG2000; the nucleic acid comprises a mRNA; and the helper lipid is selected from 1,2-dioleoyl-3- trimethylammonium- 5 170450739.1propane (DOTAP), dimethyl di octadecyl ammonium (DDAB), 1,2- dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), DSPC, 1,2-dimyristoyl-sn-glycero-3- phosphate (14PA), and 1- stearoy1-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (18PG).

[0035] In certain aspects, the nanoparticle comprises: a combined molar percentage of DLin-MC3-DMA and helper lipid ranging from about 20% to about 80%; a weight ratio of cholesterol to DMG-PEG2000 ranging from about 10 to about 500; a weight ratio of DLin- MC3- DMA to helper lipid ranging from about 1 to about 200; and a molar ratio of chargeable groups in the ionizable lipid to phosphate groups in mRNA (N / P ratio) ranging from about 4 to about 12.

[0036] In particular aspects, the nanoparticle comprises: (a) about 30 molar % DOPE, about 30 molar % DLin-MC3-DMA, about 40 molar % cholesterol, about 0.40 molar % DMG- PEG2000, and a N / P ratio of about 4; (b) about 7 molar % DSPC, about 70 molar % DLin-MC3- DMA, about 20 molar % cholesterol, about 0.04 molar % DMG-PEG2000, and a N / P ratio of about 4; or (c) about 5 molar % 18PG, about 55 molar % DLin-MC3-DMA, about 40 molar % cholesterol, about 0.40 molar % DMG-PEG2000, and a N / P ratio of about 12.

[0037] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0038] Definitions

[0039] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0040] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, recitation of “a cell”, for example, includes a plurality of the cells of the same type. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description 6 170450739.1and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0041] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / - 20%, + / - 10%, + / - 5%, + / - 1%, or + / - 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude within 5-fold, and also within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0042] As used herein, the terms “agent” or “therapeutic agent” are meant to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other medical condition. The term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, antibody fragments bearing epitope recognition sites, such as Fab, Fab’, F(ab’)2fragments, Fv fragments, single chain antibodies, antibody mimetics (such as DARPins, affibody molecules, affilins, affitins, anticalins, avimers, fynomers, Kunitz domain peptides and monobodies), peptoids, aptamers; hormones, oligonucleotides, enzymes, peptides organic or inorganic molecules, natural or synthetic compounds and the like. An agent can be assayed in accordance with the methods of the disclosure at any stage during clinical trials, during pre-trial testing, or following FDA-approval.

[0043] The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab′)2fragments, Fab′ fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” 7 170450739.1should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. These antigen binding molecules can be incorporated into the LNPs and specifically target a cell of interest, e.g. a tumor cell.

[0044] “Aptamers” are DNA or RNA molecules that have been selected from random pools based on their ability to bind other molecules. The aptamer binds specifically to a target molecule wherein the nucleic acid molecule has sequence that comprises a sequence recognized by the target molecule in its natural setting. Alternately, an aptamer can be a nucleic acid molecule that binds to a target molecule wherein the target molecule does not naturally bind to a nucleic acid. The target molecule can be any molecule of interest. For example, the aptamer can be used to bind to a ligand-binding domain of a protein, thereby preventing interaction of the naturally occurring ligand with the protein. This is a non-limiting example and those in the art will recognize that other embodiments can be readily generated using techniques generally known in the art (see, e.g., Gold et al., Annu. Rev. Biochem. 64:763, 1995; Brody and Gold, J. Biotechnol. 74:5, 2000; Sun, Curr. Opin. Mol. Ther. 2:100, 2000; Kusser, J. Biotechnol. 74:27, 2000; Hermann and Patel, Science 287:820, 2000; and Jayasena, Clinical Chem. 45:1628, 1999).

[0045] As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements--or, as appropriate, equivalents thereof--and that other elements can be included and still fall within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0046] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0047] The term “encapsulation efficiency” as used herein refers to the percentage of nucleic acid in the lipid nanoparticles that is not degraded after exposure to serum or a nuclease assay that would significantly degrade free nucleic acids.

[0048] The term “fully encapsulated” as used herein indicates that the nucleic acid in the particles is not significantly degraded after exposure to serum or a nuclease assay that would 8 170450739.1significantly degrade free nucleic acids. In a fully encapsulated system, preferably less than 25% of particle nucleic acid is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than 10% and most preferably less than 5% of the particle nucleic acid is degraded. Fully encapsulated also indicates that the particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0049] The term “lipid nanoparticle” refers to a nanoparticle that includes lipids and that is stable and dispersible in aqueous media. In exemplary embodiments, lipid nanoparticles may be from 10 nm to 500 nm in diameter, e.g., from 70 nm to 120 nm.

[0050] As used herein, the term “microparticle” refers to a small particle or particulate system, generally larger than about one micrometer (1 μm) in diameter and can be used to describe both microcapsules and microspheres.

[0051] As used herein, the term “nanoparticle” refers to a particle having one or a plurality of components, the particle having any one structural feature on a scale of less than about 1000 nm that exhibits novel properties as compared to a bulk sample of the same material or component materials. Routinely, nanoparticles have any one structural feature on a scale of less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm or less than about 100 nm. In exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-500 nm. In other exemplary embodiments, a nanoparticle is a particle having one or more dimensions of the order of about 10-1000 nm. A spherical nanoparticle would have a diameter, for example, of between 10-100 nm or 10-1000 nm.

[0052] A nanoparticle most often behaves as a unit in terms of its physical or biophysical properties, e.g., transport. It is noted that novel properties that differentiate nanoparticles from the corresponding bulk material typically develop at a size scale of under 1000 nm, or at a size of under 500 nm, but nanoparticles can be of a larger size, for example, for particles that are oblong, tubular, and the like. The size at which materials display different properties as compared to the bulk material is material-dependent and can be seen for many materials much larger in size than 100 nm and even for some materials larger in size than 1000 nm. Nanoparticles can be employed in a variety of drug delivery technologies (e.g., nucleic acid drug delivery technologies) and can be employed for various purposes including, but not limited to, controlled drug delivery, protection of the drugs from degradation, and protection of the body from the toxic effects of the drugs. 9 170450739.1

[0053] The term “nucleic acid” refers to a molecule of two or more nucleotides or alternative nucleotides. The term, “nucleotide” refers to a nucleoside including a phosphate group. The term “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a “nucleobase”). Examples of nucleic acids include but are not limited to DNA, RNA, tRNA (transfer RNA), mRNA (messenger RNA), siRNA (small interfering RNA), miRNA (micro RNA), shRNA (short hairpin RNA), ncRNA (non-coding RNA), aptamers, ribozymes, and shorter oligonucleotide sequences of any of the foregoing. Alterations of the base, sugar, and phosphate moiety of a nucleotide are encompassed by this definition.

[0054] The term also applies to nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes: a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and / or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.

[0055] The nucleic acid sequences may be “chimeric,” that is, composed of different regions. In the context of this disclosure “chimeric” compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), PNA region(s) etc. Each chemical region is made up of at least one monomer unit, i.e., a nucleotide. These sequences typically comprise at least one region wherein the sequence is modified in order to exhibit one or more desired properties.

[0056] As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise. 10 170450739.1

[0057] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0058] The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the disclosure find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.

[0059] As used herein, a “pharmaceutically acceptable” component / carrier etc. is one that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0060] “Treatment” is an intervention performed with the intention of preventing the development or altering the pathology or symptoms of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. “Treatment” may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. As defined herein, a “therapeutically effective” amount of a compound or agent (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the disclosure can include a single treatment or a series of treatments.

[0061] Ranges: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For 11 170450739.1example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0063] FIG. 1 is a schematic illustration of multi-step screening and optimization of protein encapsulated in lipid nanoparticles.

[0064] FIG. 2 is a series of heatmaps demonstrating the gene editing efficiency of LNPs in HEK Ai9 cell line via high-throughput screening platform after 24 hr incubation (n = 3) The gene editing efficiency is quantified with percentage of tdTom expression acquired through flow cytometry.

[0065] FIG. 3 is a plot showing that the formulations from each helper lipid series were selected based on transfection efficiency in HEK Ai9 cell line.

[0066] FIG.4 is a graph demonstrating the gene editing efficiency of top 50 LNPs on HEK Ai9 cells with 6 different helper lipids.

[0067] FIG. 5 is a graph demonstrating the gene editing efficiency of spleen in Ai9 mice at 5 days after i.v. injection of LNP with 50µg of Cre recombinase for in vivo cluster-mode screening with top 50 formulations.

[0068] FIG.6 is a graph showing the gene editing efficiency delivered by top 15 individual formulations in CD45+splenocytes on day 5 after i.v. injections were evaluated via FACS analysis (% of tdTom+cells).

[0069] FIGS. 7A-7D are a series of plots showing the results from FACS used to quantify the percentage of tdTom+FIG. 7A: T cells (CD45+CD3+cells), FIG. 7B: B cells (CD45+CD19+cells) FIG. 7C: dendritic cells (CD45+CD11c+cells) and FIG. 7D: macrophages (CD45+CD11b+CD11c- cells) in the spleen of in Ai9 mice.

[0070] FIG. 8 is a series of pie charts showing the composition of top four individual formulations. 12 170450739.1

[0071] FIG. 9 is a schematic illustration of multi-step screening and optimization of protein encapsulated in lipid nanoparticles (LNP). The schematic shows library screening of LNP for gene-editing protein delivery. LNP library design using DLin-MC3-DMA as the ionizable lipid, cholesterol, and DMG-PEG2000, with six helper lipids at varied ratios. In vitro screening in HEK Ai9 cells will be used to identify top-performing formulations. Top-performing LNPs were clustered by helper lipid charge into 8 groups (5 formulations per group) for further evaluation. The top formulation was selected and evaluated for gene knockout efficiency using therapeutic targets (e.g., CCR5, PD-1) in vivo.

[0072] FIG. 10 (includes FIGS. 10A-10D) shows high throughput screening for RNP LNPS with HEK Ai9 cells. FIG. 10A: Gene editing efficiency of 486 RNP LNPs was evaluated after 48-hour incubation by quantifying the percentage of tdTom+ cells using flow cytometry (n=3). The gating strategy is shown in FIG. 16. FIG. 10B. The top 50 formulations were selected based on tdTom+expression in HEK Ai9 cells, with CRISPR-Max as the control LNP reference. c,SHAP summary plots of feature impact on model output for RNP LNP machine learning models. Dots represent unique LNPs, with color indicating the relative feature value for each LNP. The location on the x-axis defines the feature value’s impact on model predictions, with values toward the right indicating enhanced gene editing outcomes. FIG. 10D: LNP library gene editing visualized in 2D space after t-stochastic neighbor embedding (t-SNE) of SHAP values. Each dot represents a unique LNP, and normalized transfection efficiency is scaled between 0 and 1 based on the minimum and maximum transfection in the dataset.

[0073] Fig.11 (includes FIGS.11A-11E): In vivo screening of T cell-targeting RNP LNP formulations. FIG.11A: In vivo cluster-based screening of RNP-loaded LNPs. Eight clusters were tested (50 µg per mouse via i.v. injection), and gene editing efficiency was assessed by measuring tdTomato+ immune cells—including T cells, B cells, CD11b+myeloid-like cells, and CD11c+dendritic cell (DC)-like cells—in the spleen via flow cytometry on day 7 post-injection. FIG.11B: Gene editing efficiency of individual formulations within the selected clusters (A, B, and C) was evaluated by quantifying tdTom+T cells (CD45+CD3+cells) in the spleen using flow cytometry on day 7 post-injection (50 µg per mouse via intravenous injection). The gating strategy is shown in FIGS. 17-18. FIG. 11C: Immune cell-type distribution of transfected tdTomato+ immune cells in the spleen following treatment with formulation F48, analyzed by flow cytometry. Cell types examined include T cells, B cells, CD11b+myeloid-like cells, and CD11c+dendritic cell (DC)-like 13 170450739.1cells. FIG.11D: Gene editing efficiency of F48 RNP-loaded LNPs in various cell types and organs (spleen, liver, and lungs) was assessed by flow cytometry on day 7 post-injection. FIG. 11E: Representative confocal images of the spleen on day 7 post-administration of F48 RNP-loaded LNPs, stained with DAPI (blue), CD45 (cyan-blue), CD3 (green), and tdTomato (red). Scale bar = 50 µm. Data represent the mean ± s.e.m. from an experiment (n = 3 (a–c) biologically independent samples, n = 7 (d) biologically independent samples).

[0074] FIG. 12 (includes FIGS.12A-12F): In vivo assessment of functional gene knock- out of CCR5 and PD-1. FIG. 12A: Schematic illustration of the mouse model used for T cell- specific gene editing and timeline of CCR5 knockout. C57BL / 6 mice were given one i.v. injection of selected LNPs loaded with 50 µg RNP (sgCCR5) per mouse on day 0 and day 7. Mice were bled on day 5 and day 10 post-injection, and spleens were extracted for analysis on day 15 (n = 4). FIG.12B: Expression of CCR5 levels on CD45+CD3+and CD45+CD3+CD4+cells in blood on day 5 and day 10, and in spleen on day 15, quantified through flow cytometry (n = 4). The gating strategy is shown in FIG. 13. FIG. 12C: Levels of CCR5 protein expression in lymphocytes from blood on day 5 and day 10, and in spleen on day 15, quantified through Western blot. Cell lysates were subjected to SDS-PAGE, followed by immunoblotting with an anti-CCR5 antibody. α-tubulin was used as a loading control. Representative blots are shown, with band intensities quantified and normalized to α-tubulin. FIG. 12D: Schematic illustration of the mouse model used for T cell- specific gene editing and timeline of PD-1 knockout. C57BL / 6 mice were first immunized with SM102 LNP loaded with mOVA (i.m.), followed by one i.v. injection of selected LNPs loaded with 50 µg RNP (sgPD-1) per mouse on day 14. Mice were sacrificed, and spleens were extracted 10 days post-injection (n = 6). FIG. 12E: Expression of PD-1 levels on CD45+CD3+and CD45+CD3+CD8+cells in spleen on day 7 post-injection, quantified through flow cytometry (n = 4). The gating strategy is shown in FIG. 23. FIG. 12F: Levels of PD-1 protein expression in lymphocytes from spleen on day 15, quantified through Western blot (n = 6). Cell lysates were subjected to SDS-PAGE, followed by immunoblotting with an anti-PD-1 antibody. α-Tubulin was used as a loading control. Representative blots are shown, with band intensities quantified and normalized to α-tubulin. Data were analyzed using unpaired t-test. NS: P > 0.05, *P  < 0.05, **P  < 0.01, ***P  < 0.001, ****P  < 0.0001.

[0075] Fig. 13 (includes FIGS. 13A-13F): High throughput screening for Cre recombinase-loaded LNPs with HEK Ai9 cells and machine learning analysis. FIG. 13A: Gene 14 170450739.1editing efficiency of 486 Cre-loaded LNPs was assessed after 48 hours by quantifying the percentage of tdTom+ cells using flow cytometry (n=3). FIG. 13B: The top 50 formulations were selected based on tdTom+ expression in HEK Ai9 cells, with DOTAP-10 serving as the reference LNP control. FIG. 13C: SHAP summary plots (left) depicting the impact of various features on machine learning model predictions. Each dot represents a unique LNP, with color indicating the relative feature value. The x-axis position denotes the feature's effect on model predictions, with values toward the right correlating with enhanced gene editing outcomes. On the right, a 2D visualization of the LNP library is generated via t-stochastic neighbor embedding (t-SNE) of SHAP values, where each dot represents a unique LNP and normalized transfection efficiency is scaled between 0 and 1. FIG.13D: In vivo cluster-based screening of Cre-loaded LNPs. Ten clusters were tested (50 µg per mouse via intravenous injection), and gene editing efficiency (tdTomato+ immune cells) in the spleen was measured by flow cytometry on day 7 post-injection. FIG. 13E: Gene editing efficiency of individual formulations within the selected cluster was evaluated by quantifying tdTomato+immune cells in the spleen via flow cytometry. FIG. 13F: Immune cell- type distribution of transfected tdTomato+immune cells in the spleen after treatment with formulation A, analyzed by flow cytometry. Cell types examined include DC-like cell, myeloid- like cells, T cells, and B cells. Data represent the mean ± s.e.m. from an experiment (n = 3 (a, b, e, f) biologically independent samples, n = 4 (d) biologically independent samples).

[0076] FIG. 14 (includes FIGS. 14A-14H): Biodistribution and cellular uptake profiles of selected LNP formulations for RNP and Cre recombinase. FIG. 14A: Biodistribution at 6, 24, and 48 h post i.v. injection of selected RNP LNPs (50 µg RNP per mouse encapsulated in cy5 labeled DMG-PEG LNP) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency across individual organs. FIG. 14B: Cellular uptake in spleen at 6, 24, and 48 h post i.v. injection of F48 RNP LNP (50 µg RNP per mouse encapsulated in Cy5-labeled DMG-PEG LNP), assessed through flow cytometry. Data are expressed as the percentage of each population (T cells, B cells, Myeloid-like cells and DC- like cells) within the Cy5+immune cells. FIG.14C: Cellular uptake of RNP LNPs in the spleen at 6, 24, and 48 hours post-i.v.injection. Mice were administered RNP LNPs containing 50 µg Cas9 protein per mouse. The percentage of Cy5⁺ cells was quantified in each immune cell populations (T cells, B cells, Myeloid-like cells and DC-like cells) in the spleen using flow cytometry. FIG. 14D: Hydrodynamic size of the selected F48 formulation with sgAi9 RNP, measured using DLS. 15 170450739.1Data represent the intensity-weighted size distribution of the nanoparticles. In particular, Data represent the mean ± s.e.m. from an experiment (n = 4 (a, b, c, e, f, g) biologically independent samples. FIG. 14E: Biodistribution at 6, 24, and 48 h post i.v. injection of selected Cre LNPs (50 µg Cre protein per mouse encapsulated in cy5 labeled DMG-PEG LNP) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency across individual organs. FIG.14F: Cellular uptake in spleen at 6, 24, and 48 h post i.v. injection of F53 Cre LNP (50 µg Cre per mouse encapsulated in Cy5-labeled DMG-PEG LNP), assessed through flow cytometry. Data are expressed as the percentage of each population (T cells, B cells, Myeloid-like cells and DC-like cells) within the Cy5+immune cells.

[0077] FIG. 15: Ethanol content impact on luciferase activity. Effect of ethanol content on luminescence signal. Luminescence intensity was measured for samples prepared with varying ethanol concentrations to assess its impact on signal output. Data represent the mean ± SEM of 3 independent replicates.

[0078] FIG.16: Gating strategy for flow cytometry of HEKAi9 high throughput screening. Initially, HEK Ai9 cells harvested were selected using SSC-A and FSC-A parameters and then singlet cells by the SSC-H–SSC-A plot. Viable cells were identified and selected based on the live / dead Fixable Aqua-A–SSC-A plot. Then the tdTom+cells were gated based on the untreated cells.

[0079] FIG. 17: Gating strategy for flow cytometry assessment of in vivo tdTom expression in spleen. Initially, lymphocytes were selected from spleen samples based on SSC-A and FSC-A parameters, followed by the exclusion of doublets using an SSC-H vs. SSC-A plot. Viable cells were identified using a Live / Dead Fixable Aqua-A vs. SSC-A plot. T cells were gated as CD3⁺, while B cells were identified as CD19⁺. Dendritic cell (DC)-like populations were defined as CD11c⁺, and macrophage-like populations were identified as CD11b⁺.

[0080] FIG. 18: Gating strategy for flow cytometry assessment of in vivo tdTom expression in liver for hepatocytes. Hepatocytes were initially selected from liver samples based on SSC-A and FSC-A parameters, followed by the exclusion of doublets using an SSC-H vs. SSC- A plot. Hepatocytes were further identified as CD45⁻CD31⁻CD326⁻cells .

[0081] FIG. 19: Cluster screening results for tdTom+ cells in hepatocytes, lung epithelial cells, and endothelial cells. In vivo cluster-based screening of RNP-loaded LNPs. Eight clusters were tested (50 µg per mouse via i.v. injection), and gene editing efficiency was assessed by 16 170450739.1measuring tdTomato+of hepatocytes, lung epithelial and endothelial cells via flow cytometry on day 7 post-injection. Data represent the mean ± s.e.m. from an experiment (n = 3 biologically independent samples).

[0082] FIG. 20: Cell composition of tdTom+cells in spleen for in cluster screening. Immune cell-type distribution of transfected tdTomato+ immune cells in the spleen following treatment with cluster DOTAP I and 18PGI, analyzed by flow cytometry. Cell types examined include T cells, B cells, CD11b+myeloid-like cells, and CD11c+dendritic cell (DC)-like cells. Data represent the mean from an experiment n = 3 biologically independent samples.

[0083] FIG. 21: Gating strategy for flow cytometry assessment of in vivo CCR5 knock out. Lymphocytes were first identified based on FSC-H vs. SSC-H parameters, followed by the selection of singlet cells using an FSC-H vs. FSC-A plot. Viable cells were gated based on a Live / Dead stain. CD45⁺ cells were identified as leukocytes, and CD3⁺ cells were gated as T cells. CD4⁺ T cells were further analyzed, and CCR5 expression was assessed in both CD3⁺ and CD4⁺ populations to examine T cell subsets.

[0084] FIG.22: Western blot image for CCR5 expression in blood and spleen. FIG.22A: Lymphocyte samples from blood at day 5 post-treatment. Lanes 1-4 represent the control group, and lanes 5-8 represent the treated group. FIG. 22B: Lymphocyte samples from blood at day 10 post-treatment. Lanes 1-4 correspond to the control group, and lanes 5-8 correspond to the treated group. FIG.22C: Lymphocyte samples from spleen at day 15 post-treatment. Each lane represents an independent biological replicate (n = 4 per group). α-Tubulin was measured as a reference protein. Differences in band intensity indicate CCR5 protein expression levels, assessing knockout efficiency over time.

[0085] FIG.23: Gating strategy for flow cytometry assessment of in vivo PD-1 knock out. Lymphocytes were first identified based on FSC-H vs. SSC-H parameters, followed by the selection of singlet cells using an FSC-H vs. FSC-A plot. Viable cells were gated based on a Live / Dead stain. CD45⁺ cells were identified as leukocytes, and CD3⁺ cells were gated as T cells. CD8⁺ T cells were further analyzed, and PD-1 expression was assessed in both CD3⁺ and CD8⁺ populations to examine T cell subsets.

[0086] FIG. 24 (includes FIGS. 24A-24B): Western blot image for PD-1 expression in spleen. FIG. 24A: Lymphocyte samples from untreated mice. FIG. 24B: Lymphocyte samples from treated mice, 10 days post-injection. Each lane represents an independent biological replicate 17 170450739.1(n = 6 per group). α-Tubulin was measured as a reference protein. Differences in band intensity indicate PD-1 protein expression levels.

[0087] FIG. 25: IVIS image for quantification of RNP LNP distribution. IVIS images showed the distribution of Cy5-labeled RNP LNPs in major organs at 6 hours, 24 hours, and 48 hours post-administration. Cy5 signal intensity indicates LNP accumulation in different organs. (n = 4 biologically independent samples)

[0088] FIG. 26: IVIS imaging for quantification of Cre LNP biodistribution. LNPs were formulated with Cy5-labeled DMG-PEG and Cy7-labeled Cre recombinase. IVIS images depict the distribution of Cre LNPs in major organs at 6 hours, 24 hours, and 48 hours post- administration. Cy5 and Cy7 signal intensities indicate LNP accumulation across different organs. (n = 4 biologically independent samples). DETAILED DESCRIPTION

[0089] Lipid nanoparticles (LNPs) have been engineered as carriers for mRNA-encoding antigens with validated biosafety profile. However, their application in protein delivery is less explored. In this study, the formulations for spleen-targeted delivery of gene-editing proteins including Cre recombinase and Cas9 were identified using this LNP screening platform. In particular, with in vitro screening with HEK293 Ai9 cell line, and further cluster-mode in vivo screening in Ai9 mice, formulations targeting the spleen for gene editing in T cells, B cells and DCs are disclosed herein.

[0090] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm. 18 170450739.1

[0091] For lipid nanoparticle nucleic acid delivery systems, the lipid shell is formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid's negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0092] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (Imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation. In some aspects, proteins, nucleic acid molecules etc., provided herein and lipids or lipid formulations provided herein form a lipid nanoparticle (LNP).

[0093] In the context of the present disclosure, a lipid nanoparticle (LNP) delivery vehicle typically serves to transport a desired therapeutic agent to a target cell or tissue. In certain embodiments, the lipid formulation encapsulates the therapeutic agent.

[0094] The manufacture of lipid nanoparticles is described in detail in the examples section. Lipid nanoparticles can also be produced (e.g., allowed to self-assemble, e.g., spontaneously) by injecting a lower alkanol solution containing lipids into an aqueous solution. Various lipids can be used to achieve desired properties, such as size, surface charge, and capacity for encapsulants. Such properties can also be influenced by the composition of the aqueous solution. Lipid nanoparticles of the invention can encapsulate a wide range of hydrophilic 19 170450739.1molecules, e.g., nucleic acids such as DNA and RNA, or alternative versions of DNA or RNA. Variations in lipid nanoparticle size can be affected by controlling process parameters. In particular, the rate at which the lower alkanol solution is injected into the aqueous solution is inversely related to the resulting lipid nanoparticle size. Similarly, minimizing variance in the rate of injection will minimize variance in lipid nanoparticle size, yielding homogeneous suspensions of lipid nanoparticles, e.g., within a single batch or among multiple batches. A precise rate of injection can be attained, e.g., through a servo pump.

[0095] Typically, the lipid nanoparticles are liposomes with a lipid bilayer surrounding an aqueous interior. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 nm and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0096] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed (e.g., osmolality or pH), the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[0097] Conventional downstream processing can be employed. For example, lipid nanoparticles can be purified or concentrated, e.g., by tangential flow filtration, dialysis, or desalting column (e.g., a PD-10 desalting column). In methods involving dialysis, the filter membrane geometry, including the area of the filter and the fiber diameter can be varied to achieve an optimal rate of filtration according to known parameters, such as lipid nanoparticle size, encapsulants size, and liquid viscosity (e.g., buffer viscosity). 20 170450739.1

[0098] In certain embodiments, the nanoparticle suspension can be exchanged with a solution containing a cryoprotectant (e.g., for long term storage in, e.g., frozen or lyophilized form). Physiologically suitable cyroprotectants for lipid nanoparticles are known in the art and include, e.g., sucrose, glucose, mannitol, glycerol, and other carbohydrates and polyalcohols. In one non-limiting example, a lipid nanoparticle solution is dialyzed against a sucrose solution (e.g., a TRIS / sucrose buffer).

[0099] A sterile filtration step may also be employed, and the membrane area, pore size and filtration force can be varied, as described above. The lipid nanoparticles described herein may be made in a sterile environment. [000100] LNP COMPOSITIONS [000101] In one aspect, a lipid nanoparticle (LNP) formulation comprises an ionizable lipid, at least one helper lipid, a PEGylated lipid, a sterol or combinations thereof. In certain embodiments, the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof. In certain embodiments, the cationic lipid comprises 1,2-dioleoyl- 3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof. In certain embodiments, the zwitterionic lipid comprises 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof. In certain embodiments, the anionic lipid comprises 1,2-dimyristoyl-sn- glycero-3-phosphate (14PA), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho (1’-rac-glycerol) (18PG) or the combination thereof. In certain embodiments, the ionizable lipid comprises DLin-MC3- DMA. In certain embodiments, the sterol comprises cholesterol. [000102] Ionizable Lipids: Other examples of ionizable lipids include 3-(didodecylamino)- N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4- tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza- octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl- 3-[(9Z,12Z)-octadeca-9,12-dien-1-yl oxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)- 21 170450739.1cholest-5-en-3-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]propan-1-amine (Octyl-CLinDMA (2R)), and (2S)-2-({8-[(3β)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2S)). [000103] PEG Modified Lipids: In certain embodiments, the PEGylated lipid comprises DMG-PEG 2000. As used herein, the terms “PEG lipid” and “PEGylated lipid refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG- modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG- CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan- 3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [000104] In certain embodiments, the PEG lipid includes, but are not limited to, 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG- DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). [000105] In certain embodiments, the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG- modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. [000106] In certain embodiments, the lipid moiety of the PEG lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. [000107] In certain embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. [000108] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species 22 170450739.1may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid. [000109] In certain embodiments, PEG lipids can be PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In some embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy- PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (—OH) groups on the lipid. In some embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In some embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an —OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment of the present invention. [000110] ADJUVANTS [000111] In some embodiments, a LNP that includes one or more lipids described herein may further include one or more adjuvants, e.g., Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide, and Pam3CSK4. [000112] THERAPEUTIC AGENTS [000113] Lipid nanoparticles may include one or more therapeutics and / or prophylactics, such as a nucleic acid. The disclosure features methods of delivering a therapeutic and / or prophylactic, such as a nucleic acid to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof comprising administering to a mammal and / or contacting a mammalian cell with a LNP including a therapeutic and / or prophylactic, such as a nucleic acid. [000114] Therapeutics and / or prophylactics include biologically active substances and are alternately referred to as “active agents”. A therapeutic and / or prophylactic may be a substance that, once delivered to a cell or organ, brings about a desirable change in the cell, organ, or other 23 170450739.1bodily tissue or system. Such species may be useful in the treatment of one or more diseases, disorders, or conditions. In certain embodiments, the therapeutic agent is a gene editing agent, e.g, Cre recombinase, CRISPR-Cas, etc. [000115] GENE EDITING AGENTS [000116] Programmable nucleases enable precise genome editing by, in some instances, introducing DNA double-strand breaks (DSBs) at specific genomic loci, thereby initiating gene editing. Generally, as embodied herein, a variety of gene editing systems can be employed to target genes, elements, or regions. In some embodiments, the gene editing agents comprise: Cre recombinases, CRISPR / Cas molecules, TALE transcriptional activators, Cas9 nucleases, nickases, transcriptional regulators, homologues, orthologs or combinations thereof. In one embodiment, the gene editing agent is a Clustered Regularly Interspaced Short Palindromic Repeated (CRISPR)- associated endonuclease, or homologues or orthologs thereof. In another embodiment, the CRISPR-associated endonuclease is Cas9 or homologues or orthologs, thereof. In some embodiments, the gene editing system comprises a CRISPR-Cas system. In some embodiments, the gene editing system comprises meganucleases. In some embodiments, the gene editing system comprises zinc finger nucleases (ZFNs). In some embodiments, the gene editing system comprises transcription activator–like effector nucleases (TALENs). These gene editing systems can be broadly classified into two categories based on their mode of DNA recognition: ZFNs, TALENs and meganucleases achieve specific DNA binding via protein-DNA interactions, whereas CRISPR-Cas systems are targeted to specific DNA sequences by a short RNA guide molecule that base-pairs directly with the target DNA and by protein-DNA interactions. Accordingly, protein targeting or nucleic acid targeting can be employed to target the DNA loc, e.g. a tumor gene. [000117] CRISPR-Cas Systems: The CRISPR-Cas system includes a gene editing complex comprising a CRISPR-associated nuclease, e.g., Cas9, and a guide RNA complementary to a target sequence situated on a DNA strand within the target sequence, e.g., in regulatory sequences or structural gene sequences. The mutation can comprise a deletion. The size of the deletion can vary from a single nucleotide base pair to about 10,000 base pairs. The mutation can comprise an insertion, that is, the addition of one or more nucleotide base pairs to the target sequence. The size of the inserted sequence also may vary, for example from about one base pair to about 300 nucleotide base pairs. The mutation can comprise a point mutation, that is, the replacement of a 24 170450739.1single nucleotide with another nucleotide. Useful point mutations are those that have functional consequences, for example, mutations that result in the conversion of an amino acid codon into a termination codon or that result in the production of a nonfunctional protein. [000118] In embodiments, the CRISPR / Cas system can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR / Cas proteins include Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9- HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. [000119] The Cas9 can be an orthologous. Six smaller Cas9 orthologues have been used and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being more than 1 kilobase shorter. [000120] In addition to the wild type and variant Cas9 endonucleases described, embodiments of the disclosure also encompass CRISPR systems including “enhanced-specificity” S. pyogenes Cas9 variants (eSpCas9), which dramatically reduce off target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in a groove that interacts with the non-target strand of DNA. This aim of this modification is to reduce interaction of Cas9 with the non-target strand, thereby encouraging re-hybridization between target and non-target strands. The effect of this modification is a requirement for more stringent Watson- Crick pairing between the gRNA and the target DNA strand, which limits off-target cleavage (Slaymaker, I.M. et al. (2015) DOI:10.1126 / science.aad5227). [000121] In certain embodiments, three variants found to have the best cleavage efficiency and fewest off-target effects: SpCas9 (K855A), SpCas9 (K810A / K1003A / R1060A) (a.k.a. eSpCas9 1.0), and SpCas9(K848A / K1003A / R1060A) (a.k.a. eSPCas9 1.1) are employed in the compositions. The disclosure is by no means limited to these variants, and also encompasses all Cas9 variants (Slaymaker, I.M. et al. Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126 / science.aad5227. Epub 2015 Dec 1). The present disclosure also includes another type of enhanced specificity Cas9 variant, “high fidelity” spCas9 variants (HF-Cas9). Examples of high 25 170450739.1fidelity variants include SpCas9-HF1 (N497A / R661A / Q695A / Q926A), SpCas9-HF2 (N497A / R661A / Q695A / Q926A / D1135E), SpCas9-HF3 (N497A / R661A / Q695A / Q926A / L169A), SpCas9-HF4 (N497A / R661A / Q695A / Q926A / Y450A). Also included are all SpCas9 variants bearing all possible single, double, triple and quadruple combinations of N497A, R661A, Q695A, Q926A or any other substitutions (Kleinstiver, B. P. et al., 2016, Nature. DOI: 10.1038 / nature16526). [000122] As used herein, the term “Cas” is meant to include all Cas molecules comprising variants, mutants, orthologues, high-fidelity variants and the like. [000123] In general, CRISPR / Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with guide RNAs. CRISPR / Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. Active DNA-targeting CRISPR-Cas systems use 2 to 4 nucleotide protospacer-adjacent motifs (PAMs) located next to target sequences for self versus non-self discrimination. ARMAN-1 has a strong ‘NGG’ PAM preference. Cas9 also employs two separate transcripts, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), for RNA-guided DNA cleavage. Putative tracrRNA was identified in the vicinity of both ARMAN-1 and ARMAN-4 CRISPR-Cas9 systems (Burstein, D. et al. New CRISPR-Cas systems from uncultivated microbes. Nature. 2017 Feb 9;542(7640):237-241. doi: 10.1038 / nature21059. Epub 2016 Dec 22). [000124] THERAPEUTIC AGENTS [000125] In certain embodiments, a therapeutic and / or prophylactic is a small molecule drug useful in the treatment of a particular disease, disorder, or condition. Examples of drugs useful in the lipid nanoparticles include, but are not limited to, antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents (e.g., actinomycin D, vincristine, vinblastine, cytosine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate and purine and pyrimidine analogs), anti-infective agents, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetalol), antihypertensive agents (e.g., clonidine and hydralazine), anti- 26 170450739.1depressants (e.g., imipramine, amitriptyline, and doxepin), anti-conversants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotic / antibacterial agents (e.g., gentamycin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, narcotics, and imaging agents. [000126] In some embodiments, a therapeutic and / or prophylactic is a cytotoxin, a radioactive ion, a chemotherapeutic, a vaccine, a compound that elicits an immune response, and / or another therapeutic and / or prophylactic. A cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, teracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol, rachelmycin (CC-1065), and analogs or homologs thereof. Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Vaccines include compounds and preparations that are capable of providing immunity against one or more conditions related to infectious diseases such as influenza, measles, human papillomavirus (HPV), rabies, meningitis, whooping cough, tetanus, plague, hepatitis, and tuberculosis and can include mRNAs encoding infectious disease derived antigens and / or epitopes. Vaccines also include compounds and preparations that direct an immune response against cancer cells and can include mRNAs encoding tumor cell derived antigens, epitopes, and / or neoepitopes. Compounds eliciting immune responses may include, but are not limited to, vaccines, corticosteroids (e.g., dexamethasone), and other species. [000127] In other embodiments, a therapeutic and / or prophylactic is a protein. Therapeutic proteins useful in the nanoparticles in the disclosure include, but are not limited to, gentamycin, amikacin, insulin, erythropoietin (EPO), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), Factor VIR, luteinizing hormone- releasing hormone (LHRH) analogs, interferons, heparin, Hepatitis B surface antigen, typhoid vaccine, and cholera vaccine. Other therapeutics and / or prophylactics include, but are not limited 27 170450739.1to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil dacarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids). [000128] POLYNUCLEOTIDES AND NUCLEIC ACIDS [000129] In some embodiments, a therapeutic agent is a polynucleotide or nucleic acid (e.g., ribonucleic acid or deoxyribonucleic acid). The term “polynucleotide”, in its broadest sense, includes any compound and / or substance that is or can be incorporated into an oligonucleotide chain. Exemplary polynucleotides for use in accordance with the present disclosure include, but are not limited to, one or more of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) including messenger RNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc. In some embodiments, a therapeutic and / or prophylactic is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer- substrate RNA (dsRNA), small hairpin RNA (shRNA), transfer RNA (tRNA), messenger RNA (mRNA), and mixtures thereof. In some embodiments, the RNA is an mRNA. [000130] In some embodiments, a therapeutic and / or prophylactic is an mRNA. An mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and my have any secondary structure or activity. In some embodiments, a polypeptide encoded by an mRNA may have a therapeutic effect when expressed in a cell. [000131] In other embodiments, a therapeutic and / or prophylactic is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a LNP including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that 28 170450739.1encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA. [000132] In some embodiments, a therapeutic and / or prophylactic is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant arts. [000133] PHARMACEUTICAL COMPOSITIONS [000134] Pharmaceutical compositions of the present disclosure comprise the lipid nanoparticles disclosed herein, a therapeutically effective amount of at least one therapeutic agent and / or one or more pharmaceutically acceptable excipient, carrier or diluent. As used herein, the term “pharmaceutically acceptable excipient, carrier or diluent” includes 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 animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In certain embodiments, the pharmaceutical compositions may further comprise at least one tissue-targeting agent, for example, peptide conjugates such as antibodies or fragments thereof, aptamers and the like. [000135] The pharmaceutical compositions may be in a form suitable for parenteral administration. Depending upon the therapeutic application, for in vivo administration, the compositions comprising anti-inflammatory lipid nanoparticles disclosed herein may be administered to a subject in need thereof intravenously, intradermally, intramuscularly, subcutaneously, sublingual, intratumorally, intracardiac, by intratracheal instillation, bronchial instillation, and / or inhalation. [000136] The pharmaceutical compositions may be in the form of a sterile injectable aqueous or suspension, which may be formulated according to known procedures. A sterile injectable preparation may also be a sterile injectable suspension in a non-toxic parenterally-acceptable buffer. In other embodiments, the pharmaceutical composition may be lyophilized resulting in the form of a dry powder, wherein the dry powder can be later reconstituted for administration as needed. Dry powder compositions may further comprise bulking agents, for example, sucrose or trehalose. 29 170450739.1[000137] Pharmaceutical liquid compositions can be nebulized by use of inert gases. Nebulized suspensions may be breathed directly from the nebulizing device or the nebulizing device can be attached to face masks tent, or intermittent positive pressure breathing machine. Furthermore, solid dosage forms may also be administered via inhalation using dry-powder inhalers. Suspension or dry powder pharmaceutical compositions can be administered orally or nasally from devices which deliver the pharmaceutical composition in an appropriate manner. [000138] The amount of a therapeutic agent that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the subject treated and the particular route of administration. For further information on routes of administration and dosage regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990. [000139] Further provided herein is a pharmaceutical kit comprising a pharmaceutical composition comprising an LNP having a therapeutically effective amount of at least one therapeutic agent. Such kits may further comprise various conventional pharmaceutical kit components such as containers comprising pharmaceutically-acceptable adjuvants, diluents or carriers, and additional containers readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit. EXAMPLES [000140] EXAMPLE 1: COMPOSITION SCREENING OF LIPID NANOPARTICLE FOR INTRACELLULAR DELIVERY OF GENE-EDITING PROTEINS [000141] The aim of this study is to optimize the formulation for gene-editing proteins including Cre recombinase and Cas9. [000142] Materials and Methods [000143] DLin-MC3-DMA was purchased from MedKoo Biosciences. DSPC, DOPE, DOTAP, DDAB, 18PG (sodium salt) and 14PA (sodium salt) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma. DMG-PEG (MW 2000) (DMG-PEG2000) was purchased from NOF America Corporation. HEK293-T Ai9 cell line was kindly provided by Prof. Kam Leong. All TAT-Cre recombinase (SCR508) was purchased from Millipore Sigma. 30 170450739.1[000144] Cell culture and high-throughput screening for transfection studies [000145] HEK293-T cell line was modified to stably express the Ai9 reporter plasmid (Addgene 22799) and cultured in DMEM supplemented with 10% FBS, 1% penicillin / streptomycin, and 1% non-essential amino acids (complete DMEM). For high throughput monolayer culture studies, HEK-293T Ai9 cells were seeded into 96-well plates at a cell density of 50,000 cells per well, immediately treated with lipid nanoparticles. The particles prepared were pipetted into DMEM medium at a final concentration of 50nM of Cre recombinase. For example, 50µl of the 200nM of Cre recombinase encapsulated LNP in PBS is added to 150µl of cell culture media. A 24 h incubation was followed to allow tdTom expression. When characterizing expression of tdTomato as the reporter, cells were digested in 0.05% of trypsin (Gibco 25200114) and quantified with Flow cytometry. [000146] High throughput LNP synthesis and characterization [000147] An organic phase was prepared by solubilizing with ethanol, a mixture of the helper lipid (DOTAP, DDAB, DOPE, DSPC, 14PA, 18PG) (Avanti), cholesterol (Sigma-Aldrich), DMG- PEG2000 (Avanti) and Dlin-MC3 DMA at a predetermined molar ratio. The aqueous phase was prepared in PBS (pH 7.4, Fisher) with Cre Recombinase. Cre recombinase was stored at −20 °C. [000148] For high-throughput screening, the ethanol phase of LNPs were prepared in a 96- well plate through liquid handler MANTIS (Formulaxtrix) through adding each individual lipid component in the well plate. The ethanol and aqueous phase are mixed with the volume ratio of 1:9. For each formulation, 16µl of ethanol phase was prepared and added with 144ul of Cre Recombinase in PBS. For in vitro screening, LNPs were directly incubated with cells without dialysis. [000149] For in vivo cluster screening, the ethanol and aqueous phases were mixed at a 9:1 ratio in small volumes with pipette. The LNPs then were dialyzed against PBS (Gibco) in a 3kDa MWCO dialysis tube (Spectrum Spectra) at 4 °C for 24 h and were stored at 4 °C before injection. The size, polydispersity index and zeta potentials of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Diameters are reported as the intensity mean average. [000150] In vivo studies for cluster screening [000151] All animal procedures were performed with ethical compliance and approval by the Johns Hopkins Institutional Animal Care and Use Committee (protocol #MO20E31). Female and 31 170450739.1male BALB / c Ai9 mice (6–8 weeks) bred in Johns Hopkins Animal Facilities and randomly grouped. Mice were generally fed a diet containing low fiber (5%), protein (20%) and fat (5–10%). The pelleted feed was supplied. Mice were supplied feed free choice, and they ate 4–5 g a day (12 g / 100 g body weight / day). Water was supplied free choice, and they usually drank 3–5 ml a day (1.5 ml / 10 g body weight / day). Water was supplied using automatic waterers. Mouse rooms were maintained at 30–70% relative humidity and a temperature of 18–26 °C (64–79 °F) with at least ten room air changes per hour. The mice were housed in standard shoebox cages with filter tops. Mice were provided with corncob as bedding. [000152] For experiments in Ai9 mice, the Cre LNP formulations were prepared as described above and injected via i.v. mouse lateral tail veiat a dose of 50 μg cre recombinase per mouse (n = 4). The LNP suspensions were concentrated to 250 μg / mL of protein by centrifugal filter unit (Amicon Ultra-2) with a MWCO of 100 kDa. After five days, mice were sacrificed, and the spleens and livers were harvested. For animal welfare monitoring, after dosing, the animals were monitored at 24 h and 48 h to ensure that they were not harmed. For animal euthanasia, mice were euthanized by CO2asphyxiation. The death of animal was verified by cervical dislocation. [000153] Cell isolation and staining for flow cytometry [000154] For high throughput in vitro studies, in preparation for the flow cytometry, cells were stained with live / dead fixable aqua dead cell stain kit (Invitrogen) for 20 min and washed twice with PBS. The percentage of tdTom+cells were quantified through Attune NxT Flow Cytometer and analyzed using FlowJo software. [000155] To quantify the tdTom+cells among different cell types in each organ for all in vivo studies, cell isolation and staining was performed, followed by flow cytometry analysis. For hepatocyte isolation, a two-step collagenase perfusion was executed. In detail, mice were anesthetized using isoflurane then fixed. Perfusion, initially using liver perfusion medium (Thermo Fisher) for 7–10 min, then switching to liver digestion medium (Thermo Fisher) for another 7– 10 min, was performed. The liver was collected on a plate containing 10 mL of liver digestion medium and cut to release the hepatocytes. The released hepatocytes were then collected and washed with ice-cold hepatocyte wash medium (Thermo Fisher) and centrifuged at 50 × g for 5 min. The supernatant was decanted, and the pellet was resuspended with an ice-cold hepatocyte wash medium. The cell suspension was passed through a 100 μm filter. The hepatocyte suspension was washed twice with ice-cold hepatocyte wash medium and once with PBS via centrifugation 32 170450739.1(50 × g) for 5 min. Afterwards, the hepatocytes were further strained through a 100 μm filter and centrifuged at 50 × g for 5 min, and cells were resuspended in 500 µL of staining buffer. The antibodies used here were Brilliant Violet 421 anti-mouse CD45 (Biolegend #103134), APC anti- mouse CD326 (Biolegend #118214), APC / Cyanine7 anti-mouse CD31 (BioLegend #102440). The dilution ratio for all antibodies listed above was 1:200 with staining buffer (ThermoFisher #00422226). Flow data were acquired on Attune NxT Flow Cytometer and analyzed using FlowJo software. [000156] For isolation and staining of spleen cells, the removed spleen was minced using a sterile blade and homogenized in 250 μL of digestion medium (45 units / μL collagenase I, 25 units / μL DNase I and 30 units / μL hyaluronidase). The spleen solution was transferred into a 15- mL tube that contained 5–10 mL of digestion medium. Next, the spleen solution was filtered using a 70 μm filter and washed once with PBS. Cells was pelleted at 300 × g for 5 min at 4 °C, and resuspended in 2 mL of red blood cell lysis buffer (BioLegend) and incubated on ice for 5 min. After incubation, 4 mL of cell staining buffer (BioLegend) was added and centrifuged again at 300 × g for 5 min. Cell pellet was washed with staining buffer for 3 times and stained with antibodies (total volume 100 μL) for 20 min in the dark at 4 °C. The stained cells were washed twice with 1 mL of PBS, then resuspended in 500 μL PBS for flow cytometry analysis. The antibodies used include Brilliant Violet 421 anti-mouse CD45 (BioLegend #103134), PerCP- Cyanine 5.5 anti-mouse CD11b (BioLegend #101228), FITC anti-mouse CD11c (BioLegend #117309), APC anti-mouse CD3 (BioLegend #100236) and PE-Cyanine 7 anti-mouse CD19 (BioLegend #159810). [000157] Results [000158] DLin-MC3-DMA was chosen as the ionizable lipid component, while DMG-PEG 2000 served as the PEGylated lipid in our study. To represent a broad spectrum of charges, six helper phospholipids were selected that have been incorporated in either experimental or FDA- approved lipid nanoparticle (LNP) formulations: the cationic lipids 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP) and dimethyldioctadecylammonium (DDAB); the zwitterionic lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC); and the anionic lipids 1,2-dimyristoyl-sn-glycero-3- phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho (1’-rac-glycerol) (18PG). 33 170450739.1Utilizing these components, an initial library of 1,080 LNP formulations was constructed. See FIG. 1. This library was systematically varied according to four parameters: (1) the combined molar percentage of DLin-MC3-DMA and the chosen helper lipid, ranging from 20% to 80%; (2) the weight ratio of cholesterol to DMG-PEG2000, spanning from 10 to 200; (3) the weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100; and (4) the weight ratio of the total lipid mass to protein, from 10 to 30. [000159] Initially, these LNPs were synthesized for in vitro screening using Cre recombinase on the HEK293 Ai9 Cre reporter cell line. In this system, successful delivery of the protein intracellularly results in the expression of tdTomato, allowing for high throughput testing via flow cytometry. As depicted in FIGS. 2 and 3, while using the same helper lipid and adjusting various parameters, significant variations in tdTomato expression levels were observed across the 486 LNPs tested. Notably, 25 formulations induced over 5% tdTomato-positive cells after treatment. [000160] The in vivo transfection efficiency of LNPs likely differs from that of traditional in vitro assay screens due to the difference between in vivo and in vitro settings and delivery barriers. To further identify the formulations that can transfect the spleen potently, to cluster-mode in vivo screening was conducted. In detail, the top 50 performing formulations from the first in vitro screening step shown above (FIG. 4) were picked and 10 clusters were generated with five formulations in each cluster. [000161] To evaluate the spleen-specific delivery efficiency of these formulations, genetically engineered tdTom reporter mice (Ai9 mice) containing a LoxP-flanked stop cassette that prevents expression of the tdTom protein were utilized. This mouse model allows the detection of the intracellular delivery of Cre recombinase. When Cre recombinase is introduced into the reporter mouse cells, it recombines the DNA at the LoxP sites to excise the stop cassette, which permits the expression offluorescent tdTom. Specifically, 10 clusters of LNPs were administrated intravenously (50 μg per mouse) and spleens were harvested on day 5 post-administration for flow cytometry analysis. Among these 10 clusters, there are three clusters (Cluster 3, 4 and 6) generated the highest tdTom+expression in CD45+lymphocytes in spleens, reaching around 15% of tdTom+expression (FIG. 5). These three clusters include the formulations containing 18PG, DDAB and 14PA, including both anionic and cationic helper lipids. [000162] To further identify the effective formulations for potent protein cargo delivery into the spleen, the delivery efficiency of 15 individual LNP formulations from the top three performing 34 170450739.1LNP clusters using Ai9 mice (50 μg Cre recombinase per mouse, i.v.) were tested. Among these, four formulations demonstrated potent spleen-targeted delivery efficiency, with high efficiency of around 15% tdTom+expression in lymphocytes within the spleen (FIG. 6). Further analysis was conducted to identify the specific immune cell types edited within the CD45+cell populations, categorized into T cells (CD3+), B cells (CD19+), macrophages (CD11b+), and dendritic cells (CD11c+). The best-performing formulation (4-53-18PG) induced substantial levels of tdTomato- positive B cells (~13%), T cells (~7%), and dendritic cells (~20%) within the spleen (FIGS. 7A- 7D). The composition of each formulation is shown in FIG. 8. Based on these findings, LNP formulations comprising ionizable lipid ranging from 10% to 90%, helper lipid from 0% to 60%, cholesterol from 10% to 60%, and PEGylated lipid from 0% to 10% demonstrated potent delivery of protein cargo to the spleen, enabling in vivo gene editing of immune cells, including T, B, and DC cells. [000163] In this study, four top-performing LNP formulations have been successfully identified capable of potent protein cargo delivery into the spleen, inducing a substantial level of editing in T, B, and DC cells through multi-step screening methods. [000164] Table 1 CodeMolar %Lipid / proteinHelper Lipid Dlin-MC3 Chol DMG-PEGwt ratio1-45-18PG 0.5 49.5 49.8 0.2 201-52-18PG 0.8 79.2 18.2 1.8 201-51-18PG 7.3 72.7 19.9 0.1 201-54-18PG 0.8 79.2 19.9 0.1 201-50-18PG 7.3 72.7 19.8 0.2 202-77-DDAB 7.3 72.7 19.8 0.2 302-50-DDAB 7.3 72.7 19.8 0.2 202-51-DDAB 7.3 72.7 19.9 0.1 202-79-DDAB 0.8 79.2 18.2 1.8 302-41-DDAB 4.5 45.5 49.5 0.5 203-47-14PA 40.0 40.0 19.8 0.2 203-48-14PA 40.0 40.0 19.9 0.1 203-50-14PA 7.3 72.7 19.8 0.2 203-54-14PA 0.8 79.2 19.9 0.1 203-42-DDAB 4.5 45.5 49.8 0.2 204-44-18PG 0.5 49.5 49.5 0.5 204-46-18PG 40.0 40.0 18.2 1.8 2035 170450739.14-47-18PG 40.0 40.0 19.8 0.2 204-49-18PG 7.3 72.7 18.2 1.8 204-53-18PG 0.8 79.2 19.8 0.2 205-24-DDAB 7.3 72.7 19.9 0.1 105-53-DDAB 0.8 79.2 19.8 0.2 205-54-DDAB 0.8 79.2 19.9 0.1 205-80-DDAB 0.8 79.2 19.8 0.2 305-81-DDAB 0.8 79.2 19.9 0.1 306-23-DDAB 7.3 72.7 19.8 0.2 106-45-DDAB 0.5 49.5 49.8 0.2 206-48-DDAB 40.0 40.0 19.9 0.1 206-69-DDAB 4.5 45.5 49.8 0.2 306-78-DDAB 7.3 72.7 19.9 0.1 307-50-DOPE 7.3 72.7 19.8 0.2 207-51-DOPE 7.3 72.7 19.9 0.1 207-53-DOPE 0.8 79.2 19.8 0.2 207-54-DOPE 0.8 79.2 19.9 0.1 207-75-DOPE 40.0 40.0 19.9 0.1 308-42-DOPE 4.5 45.5 49.8 0.2 208-44-DOPE 0.5 49.5 49.5 0.5 208-45-DOPE 0.5 49.5 49.8 0.2 208-48-DOPE 40.0 40.0 19.9 0.1 208-78-DOPE 7.3 72.7 19.9 0.1 309-50-DOTAP 7.3 72.7 19.8 0.2 209-51-DOTAP 7.3 72.7 19.9 0.1 209-53-DOTAP 0.8 79.2 19.8 0.2 209-54-DOTAP 0.8 79.2 19.9 0.1 209-65-DOTAP 25.0 25.0 49.5 0.5 3010-53-DSPC 0.8 79.2 19.8 0.2 2010-54-DSPC 0.8 79.2 19.9 0.1 2010-44-DDAB 0.5 49.5 49.5 0.5 2010-48-18PG 40.0 40.0 19.9 0.1 2010-78-18PG 7.3 72.7 19.9 0.1 30[000165] Example 2: [000166] In this study, we leveraged our high-throughput, multi-step screening platform to optimize LNPs for gene-editing protein delivery. By screening 486 formulations in vitro, we identified top candidates based on gene-editing efficiency in HEK Ai9 cells and employed a cluster-mode screening approach for in vivo evaluation in Ai9 mice, leading to the discovery of a 36 170450739.1spleen-tropic formulation with a preference for T cell gene editing. To validate its therapeutic potential, we achieved knockout of CCR5 and PD-1 in splenic T cells using LNP-encapsulated RNPs, highlighting applications in HIV resistance and cancer immunotherapy. Further investigations into biodistribution and cellular uptake provided mechanistic insights into in vivo protein delivery. [000167] In vitro screening of LNP mediated RNP delivery using HEK Ai9 Cells [000168] We designed a library of 486 unique LNP formulations by systematically varying lipid compositions while maintaining particle stability. This library incorporated six different helper lipids—DOTAP (A), DDAB (B), DOPE (C), DSPC (D), 14 PA (E), and 18 PG (F)— alongside cholesterol, DMG-PEG2000, and DLin-MC3-DMA, the ionizable lipid used in the FDA-approved Onpattro formulation. We have also optimized the aqueous and lipid phase mixing volume ratio to achieve better protein activities (fig. 15). These variations generated a diverse range of LNPs for optimizing RNP delivery. To assess their delivery efficiency, we encapsulated ribonucleoproteins (RNPs) and conducted high-throughput screening (HTS) in HEK Ai9 cells. Gene-editing efficiency was quantified using flow cytometry by measuring tdTomato fluorescence, which is activated upon successful deletion of the stop sequence. [000169] As shown in FIGS. 10A-10B, the delivery efficiency of the tested formulations varied significantly. The most potent formulation achieved approximately 13% tdTomato-positive cells following treatment, demonstrating robust gene-editing efficiency. Notably, formulations containing the cationic helper lipids DDAB and DOTAP yielded the highest number of successful edits, suggesting that positively charged lipids enhance RNP delivery, likely by promoting stronger complexation with the slightly negatively charged RNPs. Interestingly, despite its negative charge, the anionic helper lipid 18 PG exhibited the highest overall gene-editing efficiency. This unexpected result may be attributed to its unique lipid tail structure or its ability to form particularly stable complexes with RNPs. In contrast, zwitterionic helper lipids such as DOPE and DSPC showed minimal editing activity, reinforcing the idea that charge interactions play a critical role in determining LNP performance for protein delivery. These findings highlight the importance of lipid composition in optimizing LNP formulations for efficient intracellular delivery of gene- editing proteins. [000170] To further analyze the HTS results, we employed a machine learning model trained on the LNP formulation data and gene editing efficiency outcomes (FIG. 9A-9D). Using SHAP 37 170450739.1values, we quantified the relative importance of each formulation feature and identified some key features. Each dot represents an individual LNP formulation, with the color corresponding to the relative value of the feature in that formulation. The position of the dot on the x-axis reflects the impact of the feature value on the model's output, where a positive x-value indicates a positive contribution to the predicted gene-editing efficiency. We have revealed that 1) formulations with a higher combined percentage of helper lipid and ionizable lipid (e.g., DLin-MC3-DMA) showed improved gene editing efficiency, likely due to enhanced particle stability and cargo endosomal escape; 2) preference for unsaturated lipid tails, as their fluidity promotes endosomal escape and intracellular delivery; 3) absence of negative charges in helper lipid, as cationic or neutral helper lipids (e.g., DOTAP, DDAB) outperformed anionic lipids, consistent with the observed trends in HTS; 4) formulations with a lower lipid-to-protein ratio demonstrated higher gene editing efficiency, suggesting that excessive lipid content may hinder RNP release or cellular uptake. [000171] Cluster-mode in vivo screening in Ai9 mice identifies T cell-targeting gene- editing LNP formulations [000172] The gene-editing efficiency of RNP loaded LNPs in vivo often differs from that observed in traditional in vitro assays due to the complexities of biological environments and additional delivery barriers. To identify the optimal formulation for T cell-targeted delivery, we selected the most effective LNPs based on in vitro transfection efficiency and tested them via intravenous injection. To streamline the in vivo screening process while reducing the number of animals, time, and cost, we employed a cluster-mode screening approach. Specifically, the top 50 formulations with the highest gene-editing efficiency in HEK Ai9 cells were first grouped into 10 clusters, each containing five formulations, for initial testing. The top-performing clusters were then selected, and the individual formulations within these clusters were further analyzed to identify the most potent ones. This method allowed for an efficient initial evaluation of in vivo performance, guiding the selection of top-performing formulations for further validation. [000173] Each pooled cluster was first tested in Ai9 mice, which express tdTomato upon successful deletion of the stop codon upstream of the tdTomato expression cassette by stop- sequence-targeting RNPs. Mice received a systemic intravenous (i.v.) injection of 50 µg sgAi9- containing RNPs per dose. As the result showed that while most clusters showed no detectable gene-editing activity in the liver and lung, they exhibited high gene-editing efficiency in the spleen, particularly in CD45⁺CD3⁺ T cells (FIG. 11A). The top two performing clusters, DOTAP I and 38 170450739.118PG I, showed up to ~6.5 % and 5% of tdtomato+CD45⁺CD3⁺ T cells, respectively, while exhibiting minimal gene-editing activity in non-lymphoid organs. Specifically, Cluster DOTAP I showed 0.2% and 0.43% gene-editing efficiency in hepatocytes and lung eptithelial cells, respectively, whereas Cluster 18PG I exhibited 0.02% and 0.11% gene-editing efficiency in these cell populations (FIG. 19). Although gene editing was observed in other immune cell populations within the spleen, T cells represented the majority of edited cells, accounting for 86% and 81% of all tdTomato⁺ CD45⁺immune cells for the top two clusters - DOTAP I cluster and 18PG I cluster (FIG. 20), respectively. This result underscores the optimized LNP formulations' strong T cell- targeting capability and their potent gene-editing activity in T cells. [000174] To further identify the most effective individual formulation for T cell-targeted delivery, we evaluated 15 formulations from the top three clusters identified in the initial screening. Each formulation was administered to Ai9 mice at a dose of 50 µg LNP-encapsulated sgAi9 RNPs. While delivery efficiency varied significantly among formulations, formulation F48 emerged as the top candidate, achieving ~ 7.8% gene-editing efficiency in T cells, followed by formulations A41 and F47, which showed ~ 7.0% and 4.8% transfection, respectively(FIG. 11B). Further analysis of the edited cell populations revealed that 67% of tdTomato-positive (tdTom+) cells were T cells (FIG. 11C), demonstrating a strong preference for T cell-specific editing. Remarkably, formulation F48 achieved this high T cell specificity with minimal background expression in other cell types within the spleen, liver, and lung. These results highlight the ability of F48 to achieve targeted gene editing in T cells while avoiding off-target effects in non-target tissues(FIG. 11D). [000175] To further validate these findings, we performed confocal microscopy imaging on spleen tissue sections from Ai9 mice treated with the optimal formulation F48 (FIG. 11E). The tissue was stained for CD45 and CD3 to identify immune cell populations. The imaging revealed bright tdTomato expression colocalized with CD45+ and CD3+ immune cells, confirming successful gene editing in T cells within the spleen. These results provide visual confirmation of the spleen-tropic gene editing pattern and the high gene-editing of F48 for T cells. [000176] Functional gene editing of CCR5 and PD-1 in C57BL / 6 mice [000177] To assess the therapeutic potential of our optimized LNP platform, we conducted functional gene editing of two clinically relevant targets, CCR5 and PD-1, in C57BL / 6 mice. CCR5 serves as a co-receptor for HIV entry, and its knockout can confer resistance to HIV 39 170450739.1infection, while PD-1 is an immune checkpoint receptor whose knockout can enhance anti-tumor immunity by reversing T cell exhaustion. [000178] We first evaluate the efficiency and durability of CCR5 knockout. F48 LNP- encapsulated sgCCR5 RNPs was administered to C57BL / 6 mice via systemic i.v. injection. The treatment regimen consisted of two doses, administered on day 0 and day 7, to enhance gene editing efficiency. Blood samples were collected on day 5 and day 10 to monitor CCR5 expression in peripheral immune cells, and spleens were harvested on day 15 for comprehensive analysis (FIG. 12A). Flow cytometry was performed on immune cells isolated from the blood (day 5 and day 10) and spleen (day15) to quantify CCR5 surface expression. The results demonstrated a significant, time-dependent reduction in CCR5 expression in T cells (CD3+and CD3+CD4+) (FIG. 12B). The F48 LNP treatment group showed an 40% reduction on day 5 and day 10 in CD4+T cells. The results were further validated through western blot analysis. The results revealed a significant decrease in CCR5 protein levels both in blood (day 5 and 10) and spleen (day 15) , consistent with the flow cytometry data (FIG. 12C, FIG.19). Compared to the PBS group, CCR5 protein levels decreased by 0.5-fold following F48 LNP treatment on day 10 and day 15.This confirmed the successful knockout of CCR5 at both the protein and cellular levels. These results demonstrate that our LNP platform can achieve efficient and durable CCR5 knockout in vivo, with potential applications in HIV resistance and other therapeutic contexts. [000179] We next evaluated the functional knockout of PD-1 in C57BL / 6 mice using our optimal LNP formulation. Given that the mouse PD-1 knockout sgRNA sequence is well- documented in the literature, we anticipated higher gene editing efficiency compared to CCR5. Mice were immunized with 8ug mOVA LNP to stimulate the PD-1 expression in spleen 14 days prior to the RNP injection. On day 14, mice were administered a single dose of 50 µg LNP- encapsulated sgPD-1 RNPs through i.v., and spleens were harvested 10-day post-injection for analysis (FIG. 12D). Flow cytometry revealed a ~70% reduction in PD-1 expression in CD45+CD3+T cells and an even more pronounced ~85% reduction in CD45+CD3+CD8+cytotoxic T cells (FIG. 12E). The reduction of PD-1 in CD8+T cells is particularly significant, as PD-1 is a key immune checkpoint receptor that suppresses T cell activity; its knockout can enhance anti- tumor immunity by reversing T cell exhaustion and restoring cytotoxic function. Western blot analysis of splenic protein lysates further confirmed a significant decrease in PD-1 protein levels, validating the knockout at the molecular level (FIG. 12F, FIG. 24). Compared to the PBS group, 40 170450739.1PD-1 protein levels decreased by 0.4-fold following F48 LNP treatment on day 10. These results demonstrate the ability of our LNP platform to achieve efficient and functional PD-1 knockout, highlighting its potential for cancer immunotherapy. [000180] Systematic screening of LNP formulations for efficient Cre recombinase delivery [000181] To demonstrate the versatility of our LNP platform, we next evaluated its ability to deliver Cre recombinase, a physiologically distinct protein compared to RNPs. Unlike RNPs, which are slightly negatively charged and have a relatively higher molecular weight (150kDa), Cre recombinase is smaller (38kDa) and has a higher isoelectric point, resulting in a more neutral and positive charge under physiological conditions. We conducted the same high-throughput screening process using the 486-formulation library and identified a significantly higher number of hits in formulations containing 18 PG and 14 PA helper lipids(FIG. 13A-13B). These anionic lipids likely facilitated better complexation with Cre recombinase due to is slightly positive charge. [000182] To further analyze these trends, we applied the same machine learning model used for RNP screening. Key findings from the screening revealed that formulations with 80% combined ionizable and helper lipid content showed higher gene editing efficiency, and we have seen the similar trend in RNP formulations (FIG. 13C). Additionally, a medium lipid-to-protein ratio of 20 achieved optimal gene editing, striking a balance between cargo loading and delivery efficiency. Reducing the PEG concentration also improved gene editing, likely by minimizing steric hindrance and enhancing cellular uptake and endosomal escape. Similar to our RNP results, formulations with unsaturated helper lipid tails (e.g., DOPE and 18PG) outperformed those with saturated tails, likely due to improved membrane fluidity and endosomal escape. These results highlight the adaptability of our LNP platform for delivering diverse protein cargoes, including Cre recombinase, while providing insights into the formulation parameters that drive efficient gene editing. [000183] To identify the most effective formulations for Cre recombinase delivery in vivo, we conducted a cluster-mode screening study using the top 50 formulations, grouped into 10 clusters (Fig.5d). The results revealed that the top 15 formulations were predominantly composed of DDAB, 18 PG, and 14 PA helper lipids, overlapping our in vitro findings. We then harvested the spleen, liver and lung, to check the gene editing efficiency of the cell populations of these major organs. Similar to the delivery of RNPs, gene editing events were primarily observed in the 41 170450739.1spleen, with ~15% of CD45+ immune cells showing tdTomato expression, indicating successful Cre-mediated recombination (FIG. 13E). [000184] Further analysis of the tdTomato-positive cell populations revealed a distinct distribution compared to RNP delivery. While RNP-mediated gene editing was largely restricted to T cells, Cre recombinase delivery resulted in a broader range of edited immune cell types, particularly B cells and CD11c+DC-like cells (FIG.13F). This suggests that Cre recombinase, due to its smaller size and neutral charge, may have greater accessibility to diverse immune cell populations within the spleen. The optimal formulation achieved efficient gene editing across multiple cell types, demonstrating the versatility of our LNP platform for delivering diverse protein cargoes. [000185] The biodistribution of top-performing formulations reveals cell type- dependent intracellular trafficking [000186] To assess the biodistribution and cellular uptake of our LNPs, we formulated the top-performing RNP-LNP (F48 LNP) and Cre-LNP (F53 LNP) with Cy5-labeled DMG-PEG and tracked their distribution using IVIS imaging at 6, 24, and 48 hours post-injection. Unexpectedly, both LNPs exhibited similar biodistribution patterns, with the majority of the signal accumulating in the liver (~60%) and less than 10% detected in the spleen across all time points (FIG.14A, 14E). The RNP-LNP had a particle size of approximately 200 nm (FIG. 14D), similar to nucleic acid- based LNPs, which may explain their comparable biodistribution. The Cre-LNP also measured around 200 nm (FIG.14H), with a polydispersity index (PDI) similar to nucleic acid-based LNPs, further supporting the observed similarity in distribution patterns. [000187] Despite the substantial accumulation of LNPs in the liver, gene-editing efficiency remained low, suggesting that efficient intracellular trafficking—including cellular uptake and endosomal escape—did not occur in liver cells. Conversely, although only a small fraction of LNPs (~10%) localized to the spleen, significant gene editing was observed, indicating that the LNPs successfully entered target cells and completed intracellular processing. [000188] Flow cytometry analysis further revealed distinct cellular uptake profiles within the spleen. RNP-LNPs were predominantly taken up by T cells, with 52% of T cells being Cy5- positive at 6 hours, followed by a gradual decline over time (FIGS. 14B-14C). In contrast, Cre- LNPs showed higher uptake in B cells (~21%) and DC-like CD11c+ cells (~52%) at 6 hours, with 42 170450739.1a similar decreasing trend over time (FIG. 14F, 14G). These results suggest that the initial uptake of LNPs by specific immune cell populations may influence subsequent gene-editing efficiency. [000189] The distinct uptake profiles of RNP-LNP and Cre-LNP highlight the importance of tailoring LNP formulations for targeted gene editing in specific immune cells. Although protein- similar accumulation levels in the liver and spleen as mRNA-LNPs, their ability to achieve spleen-targeted delivery may be attributed to the drastic changes in LNP physical properties induced by the protein cargo, particularly its enhanced endosomal escape capacity. This property likely makes protein-LNPs more suitable for immune cell uptake and processing compared to liver cells, further emphasizing the need for cargo-specific LNP design in therapeutic applications. [000190] Discussion [000191] While LNP-mediated delivery of RNP for in vivo gene editing has been reported, most studies have primarily targeted the liver and lungs []. A T cell-targeting LNP for RNP delivery has yet to be developed, representing a critical gap in the field. Furthermore, existing studies often repurpose LNP formulations originally designed for neucleic acids, lacking a methodical optimization of LNP compositions for protein delivery. This is a significant limitation, as proteins differ fundamentally from nucleic acids in terms of size, charge, and stability, necessitating tailored delivery systems. This study addresses these gaps by systematically exploring how LNP composition can be optimized for protein delivery, with a particular focus on enhancing T cell- targeted gene-editing efficiency. By screening a diverse library of LNP formulations and employing a machine learning-guided approach, we identified key lipid components and formulation parameters that enhance protein delivery efficiency. Our findings demonstrate that LNPs can be engineered to achieve efficient delivery of gene-editing proteins, such as RNPs and Cre recombinase, to T cells in vivo, offering a promising approach for novel immunotherapies and advancing precise gene editing strategies for treating genetic and immune-related diseases. [000192] Our biodistribution data revealed that the majority of LNPs accumulated in the liver after intravenous administration, consistent with previous reports of LNP-mediated nucleic acid delivery. However, despite this liver tropism in terms of in vivo LNP distribution, the gene-editing events were predominantly observed in T cells within the spleen. This discrepancy can be attributed to the distinct endosomal environment of T cells, which may facilitate more efficient protein delivery and endosomal escape compared to hepatocytes. T cells exhibit a more acidic 43 170450739.1endosomal pH and a higher density of endosomal escape machinery, both of which could enhance the proton sponge effect of ionizable lipids, leading to more effective cytosolic release of RNPs. Additionally, the highly vascularized structure and dense population of immune cells in the spleen may promote localized uptake and gene editing in T cells, even though the overall LNP accumulation in the spleen is lower compared to the liver. These findings highlight the importance of tailoring delivery systems to exploit the unique biological features of target cell types and suggest that LNPs can be further optimized to enhance gene-editing efficiency by taking advantage of cell-specific endosomal environments and immune cell properties. [000193] We have previously demonstrated the use of machine learning to optimize LNP formulations for nucleic acid delivery. In this study, we extend this approach to protein delivery, marking the first application of ML analysis to systematically evaluate LNP formulations for gene- editing proteins. The results revealed that the feature importance trends varied between different protein cargoes. For example, a lower percentage of PEGylated lipids was crucial for achieving high gene-editing efficiency with Cre recombinase, but this feature was less impactful for RNP delivery. This difference likely stems from the distinct physicochemical properties of the two proteins–such as size, charge, and stability–which influence their interactions with LNP components. Additionally, the ML model highlighted that unsaturated lipid tails and higher ionizable lipid content were consistently important across both cargo types, underscoring their role in enhancing endosomal escape and particle stability. These findings illustrate that ML analysis can provide cargo-specific insights into formulation optimization, enabling the design of tailored LNPs for diverse therapeutic applications. Leveraging these insights, we can further refine LNP formulations to achieve more efficient gene-editing outcomes for a wide range of protein cargoes. [000194] Although this study did not include therapeutic models, the successful functional knockout of CCR5 and PD-1 in T cells serves as a robust proof-of-concept for the efficacy of our RNP-encapsulated delivery system. The CCR5 knockout demonstrates the potential of this approach for HIV prevention, as CCR5 serves as a co-receptor for HIV entry and its deletion confers resistance to HIV infection. Similarly, the PD-1 knockout showcases the potential of this system for tumor immunotherapy, as PD-1 is a key immune checkpoint receptor that suppresses T cell activity; its deletion can enhance anti-tumor immunity by reversing T cell exhaustion, thereby boosting the adaptive immune responses against tumors. These findings underscore the promising usage of our formulation in achieving precise and efficient gene editing of T cells, with significant 44 170450739.1implications for both infectious disease and cancer immunotherapy. Further investigations could evaluate the therapeutic efficacy of CCR5 and PD-1 knockout in preclinical models, as well as the potential for targeting other immune checkpoints or disease-related genes in T cells, which could open new avenues for precision medicine in immune modulation and immunotherapy. [000195] In summary, we demonstrated the potential of LNP-encapsulated RNPs for efficient and precise gene editing in immune cells, particularly T cells in the spleen. By systematically optimizing LNP formulations along with a machine learning-guided approach, we identified key lipid components and formulation parameters that enhance the in vivo delivery efficiency of gene- editing proteins. Our findings highlight the importance of spleen-targeting and the unique endosomal environment of T cells in achieving efficient gene editing. The successful functional knockout of CCR5 and PD-1 serves as a strong proof-of-concept for therapeutic potential of our platform. These insights pave the way for future studies aimed at further refining LNP formulations, assessing therapeutic efficacy in preclinical models, and expanding the applications of this platform to other immune cell types and disease targets. Overall, our work contributes to the advancement of protein delivery and gene editing technologies, offering a versatile and efficient platform for immune cell-specific therapies with broad implications for both infectious disease and cancer immunotherapy. [000196] Methods [000197] Materials [000198] DLin-MC3-DMA was obtained from MedKoo Biosciences. DSPC, DOPE, 18PG, 14PA, DOTAP, and DMG-PEG-2000 were from Avanti Polar Lipids, and cholesterol was from Sigma-Aldrich. The Cre recombinase (SCR508) was purchased from Sigma-Aldrich, and CRISPR Cas9 (CAS-EE109) was purchased from Kactus. Lipofectamine™ CRISPRMAX™(CMAX00015) was purchased from Thermo Fisher. The HEK Ai9 cells, a tdTomato reporter cell line, were gift from Dr. Kam Leong Lab. [000199] Animals and cell culture [000200] All animal procedures were conducted in accordance with protocols approved by the Johns Hopkins Institutional Animal Care and Use Committee (Protocol #MO23E31). C57BL / 6 mice (male and female), aged 6–8 weeks, were obtained from the Jackson Laboratory. Ai9 mice (male and female), 6–8 weeks old, were bred in the Johns Hopkins Animal Facilities and assigned 45 170450739.1randomly to groups in the studies. The mice had free access to pelleted feed and water, with the feed typically containing 5% fiber, 20% protein, and 5–10% fat. On average, the mice consumed 4–5 g of pelleted feed (120 g per kg body weight) and drank 3–5 mL of water (150 mL per kg body weight) daily. The temperature in the mouse rooms was maintained between 18–26 °C (64– 79 °F) with 30–70% relative humidity, ensuring at least 10 air changes per hour. The mice were housed in standard shoebox cages with corncob bedding. [000201] For experiments in Ai9 mice and C57BL / 6 mice studies, the LNPs were given through i.v. (lateral tail vein) injection at a predetermined dose per mouse. The LNP suspensions were concentrated to 250 µg / mL for i.v. injection of protein by an Amicon Ultra-2 centrifugal filter(Sigma Millipore) unit with an MWCO of 100 kDa., the RNP and Cre LNP formulations were prepared as described above and administered via i.m. or i.v. injections at an mRNA dose of 50 μg per mouse. [000202] LNP screening and production [000203] LNPs were prepared by mixing an organic phase containing the lipid components with an aqueous phase containing the protein cargo. The ethanol phase was prepared by dissolving a mixture of ionizable lipid (DLin-MC3 DMA), cholesterol, DMG-PEG2000, and a helper lipid (DOTAP, DDAB, DOPE, DSPC, 14PA, or 18PG) at predetermined ratios in ethanol. The ethanol phase for the 486 formulations was synthesized in a 96-well plate using a liquid handling robot (Mantis Liquid Dispenser). LNPs were then prepared by mixing the aqueous phase into the ethanol phase at a 9:1 volumetric ratio. For RNP LNPs, the aqueous phase was prepared by incubating Cas9 protein and sgRNA at a 1:1 molar ratio for 10 minutes in PBS at room temperature. For Cre LNPs, Cre recombinase was dissolved directly in PBS. For high-throughput in vitro screening, LNPs were dosed without dialysis. For in vivo screening, LNPs were formulated in 1.5 mL microcentrifuge tubes through pipette mixing. When synthesized for cluster mode, LNPs were combined pre-dialysis and dialyzed using a 100-kDa MWCO cassette against PBS for 6 hours at 4 °C and stored at 4 °C before injection. LNP size and polydispersity index (PDI) were determined using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Reported diameters are represented as intensity mean averages. [000204] Tissue and blood processing and cell isolation 46 170450739.1[000205] For isolation of cells from the liver, lungs, and spleen in the Ai9 mouse experiments, the harvested tissues were placed onto 40-μm cell strainers and digested mechanically with the back of a 3-mL syringe plunger in PBS. The cells were pelleted at 300 ×g for 5 min at 4 °C, followed by resuspension in the ACK lysis buffer and incubation at room temperature for 5 min to lyse red blood cells. Cells were then pelleted by centrifugation at 300 ×g for 5 min at 4 °C, washed with RPMI 1640 medium and pelleted twice before staining for flow cytometry. All steps were performed protected from light. [000206] For isolation of immune cells from blood in C57BL / 6 mice, the 200ul of blood was collected in EDTA tubes.The blood is then transferred to 15 mL tube containing 5 mL of ACK lysis buffer, vortex shortly and incubate at RT for 3 min. Cells were then pelleted by centrifugation at 300 ×g for 5 min in at 4 °C, washed with RPMI 1640 medium twice before staining for flow cytometry and cell lysis for westernblot. The process for isolating immune cells from the spleen was identical to that described for the spleen in Ai9 mice. Briefly, spleens were mechanically dissociated, red blood cells were lysed using ACK buffer, and cells were washed and pelleted before further analysis. [000207] Antibodies and staining for flow cytometry [000208] Antibody panels are provided in Supplementary Table 1. All antibodies were diluted at a ratio of 1:100 before use. LIVE / DEAD fixable dead cell stain kits were used to determine the viability of cells. [000209] Isolated cells from the tissues, as described in the previous section, were resuspended and pelleted in 100 µL of antibodies diluted in flow cytometry staining buffer obtained from eBioscience™. The cells were then incubated on ice in the dark for 1 h. After the incubation period, the stained cells were washed twice with PBS and subsequently resuspended in 200 µL of eBioscience™ flow cytometry staining buffer for flow cytometry analysis. Flow data was acquired using an Attune NXT flow cytometer and analyzed with FlowJo software v.10. [000210] Western blot [000211] Cells were harvested and lysed using RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Thermo Fisher Scientific) on ice for 30 minutes. The lysates were centrifuged at 12,000 × g for 15 minutes at 4°C, and the supernatants were collected 47 170450739.1for protein quantification using the BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of total protein (15 µg per lane) were mixed with 4X LDS Sample Buffer (Invitrogen) and 10X Sample Reducing Agent(Invitrogen), boiled at 80°C for 10 minutes. Samples were loaded onto a Bolt 4–12% Bis-Tris Plus Gel (Thermo Fisher Scientific) and run in Bolt MES Running Buffer at 200V for 35 minutes. Proteins were transferred to a PVDF membrane (Thermo Fisher Scientific) using the iBlot 3 Dry Transfer System. Membranes were blocked in 5% non-fat dry milk in PBST (PBS with 0.1% Tween-20) for 1 hour at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: anti-CCR5 antibody (1:1000 dilution, Invitrogen, PA5-78949), anti-PD-1 antibody (1:1000 dilution, Invitrogen, PA5-20350), and anti- α-tubulin antibody (1:4000 dilution, Invitrogen, PA5-19489). [000212] After washing with PBST, membranes were incubated with HRP-conjugated secondary antibodies (goat anti-rabbit, 1:10,000 dilution, Invitrogen 32460) for 1 hour at room temperature. Membranes were then washed and developed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Scientific) according to the manufacturer’s protocol. Chemiluminescent signals were detected using a Bio-Rad ChemiDoc Imaging System. Band intensities were quantified using ImageJ software (NIH), and relative protein expression levels were normalized to α-tubulin. [000213] Confocal Microscopy [000214] Mouse spleens were harvested and embedded in OCT compound (Tissue-Tek) for cryosectioning. Cryosections were cut at a thickness of 10 µm using a cryostat and mounted onto glass slides. Sections were then permeabilized with 1.0 % Triton X-100 in PBS for 1 hr. For immunostaining, sections were blocked in 10% normal donkey serum in PBS for 10 minutes at room temperature, then incubated overnight at 4°C with primary antibodies: anti-CD3 (1:200 dilution, Abcam, ab135372) and anti-CD45 (1:200, GeneTex, GT0014). After washing 3 × 5 minutes in PBS, sections were incubated for 1 hour at room temperature with secondary antibodies: Cy3-conjugated donkey anti-mouse (1:1000 dilution, Jackson ImmunoResearch Laboratories Inc., 715-165-151) and Cy5-conjugated donkey anti-rabbit (1:1000 dilution, Jackson ImmunoResearch Laboratories Inc., 711-175-152). Nuclei were counterstained with DAPI for 15 minutes at room temperature. Sections were mounted with fluorescence mounting medium (e.g., ProLong™ Gold Antifade, Thermo Fisher Scientific) and coverslipped. 48 170450739.1[000215] Imaging was performed using a Zeiss Apotome 3 microscope with 20X and 63X objective lens, and images were acquired using Zeiss Zen software. Image processing and analysis were conducted using ImageJ software. [000216] Statistics [000217] Unpaired t-tests were performed when comparing two groups. Statistical analysis was performed using Microsoft Excel and Prism 10 (GraphPad). A difference was considered significant if P < 0.05 (*P  < 0.05, **P  < 0.01, ***P  < 0.001, ****P  < 0.0001). [000218] Supplementary Table 1. Anti-mouse antibodies used in flow cytometry panels. Marker, fluorophore, catalogue number, source, and concentration are indicated. Antigen Fluorophore Catalogue # Source Concentration CD3 APC 100236 Biolegend 1:100 dilution Live / Dead Live / Dead Fix Aqua L34957 Thermo Fisher 1:1000 dilution CD45 FITC 103108 Biolegend 1:250 dilution CD31 APC-Cy7 102440 Biolegend 1:100 dilution CD45 Brilliant Violet 421 103134 Biolegend 1:250 dilution CD19 PE-Cy7 25019382 Thermo Fisher 1:200 dilution CD11b PerCP-Cy5.5 101228 Biolegend 1:200 dilution CD11c FITC 117306 Biolegend 1:200 dilution CD3 PE 100206 Biolegend 1:200 dilution CD8 FITC 100706 Biolegend 1:200 dilution CD4 PerCP-Cy5.5 100540 Biolegend 1:200 dilution Fixable Live / Dead Live / Dead Fix Near IR (780) L10119 Thermo Fisher 1:1000 dilution CD3 APC 100236 Biolegend 1:200 dilution CD326 APC 118214 Biolegend 1:100 dilution CCR5 PE 107006 Biolegend 1:50 dilution PD-1 APC 135210 Biolegend 1:100 dilution 49 170450739.1[000219] Supplementary Table 2. Top 15 RNP LNP Formulations for in vivo individual formulation testing. Molar Ratio tionhelpe(%) FormularlipidDMG- weight ratio Dlin Helper Cholesterol PEG 3 DOTAP 10.0 10.0 79.6 0.4 10 11 DOTAP 25.0 25.0 49.5 0.5 10 24 DOTAP 72.7 7.3 19.9 0.1 10 41 DOTAP 45.5 4.5 49.5 0.5 20 77 DOTAP 72.7 7.3 19.8 0.2 30 21 18PG 40.0 40.0 19.9 0.1 10 39 18PG 25.0 25.0 49.8 0.2 20 47 18PG 40.0 40.0 19.8 0.2 20 48 18PG 40.0 40.0 19.9 0.1 20 74 18PG 40.0 40.0 19.8 0.2 30 12 DOPE 25.0 25.0 49.8 0.2 10.0 23 DOPE 72.7 7.3 19.8 0.2 10.0 46 DOPE 40.0 40.0 18.2 1.8 20.0 48 DOPE 40.0 40.0 19.9 0.1 20.0 78 DOPE 72.7 7.3 19.9 0.1 30.0 50 170450739.1[000221] Supplementary Table 3. Top 15 Cre LNP Formulations for in vivo individual formulation testing. Molar Ratio (% ormulationhel) FperlipidDMG- weight ratio Dlin Helper Cholesterol PEG 47 40.0 40.0 19.8 0.2 204840.0 40.0 19.9 0.1 20 50 14PA 72.7 7.3 19.8 0.2 20 54 14PA 79.2 0.8 19.9 0.1 20 44 18PG 49.5 0.5 49.5 0.5 20 46 18PG 40.0 40.0 18.2 1.8 20 47 18PG 40.0 40.0 19.8 0.2 20 49 18PG 72.7 7.3 18.2 1.8 20 53 18PG 79.2 0.8 19.8 0.2 20 23 DDAB 72.7 7.3 19.8 0.2 10 45 DDAB 49.5 0.5 49.8 0.2 20 48 DDAB 40.0 40.0 19.9 0.1 20 69 DDAB 45.5 4.5 49.8 0.2 30 78 DDAB 72.7 7.3 19.9 0.1 30 42 DDAB 45.5 4.5 49.8 0.2 20 OTHER EMBODIMENTS [000222] From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. [000223] All citations to sequences, patents and publications in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their disclosure. 51 170450739.1

Claims

What is claimed:

1. A lipid nanoparticle (LNP) formulation comprising: an ionizable lipid, at least one helper lipid, a polyethylene glycol (PEG)-modified lipid, a sterol, and at least one bioactive protein agent.

2. A lipid nanoparticle (LNP) formulation comprising: an ionizable lipid, at least one helper lipid, a polyethylene glycol (PEG)-modified lipid, a sterol, and at least one gene editing agent.

3. The lipid nanoparticle (LNP) formulation of claim 2 wherein the gene editing agent comprises a single strand guide RNA which targets a specific nucleic acid sequence.

4. The lipid nanoparticle (LNP) formulation of claim 2 wherein the gene editing agent is CRISPR-Cas9 system.

5. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 4 wherein the LNP further comprises a therapeutic agent.

6. The lipid nanoparticle (LNP) formulation of claim 5 wherein the therapeutic agent is encapsulated by the LNP.

7. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 6 wherein the LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 5 to about 50.

8. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 7, wherein LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

9. The lipid nanoparticle (LNP)formulation of any one of claims 1 to 7, wherein LNP formulation comprises a weight ratio of total lipid mass to therapeutic agent ranges from about 10 to about 30.

10. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 9, wherein at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof. 52 170450739.

111. The lipid nanoparticle (LNP) formulation of claims 1 to 10 wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), or the combination thereof.

12. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 11 wherein the formulation comprises an zwitterionic lipid comprising 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), or the combination thereof.

13. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 12 wherein the formulation comprises an anionic lipid that comprises 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho (1’-rac-glycerol) (18PG), or the combination thereof.

14. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 13 wherein the ionizable lipid comprises DLin-MC3-DMA.

15. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 14 wherein the polyethylene glycol (PEG)-modified lipid comprises DMG-PEG 2000.

16. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 15 wherein the sterol comprises cholesterol.

17. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 16 wherein the LNP formulation comprises a combined molar percentage of DLin-MC3-DMA and at least one helper lipid in a range from about 10 w / w% to about 90 w / w%, 18. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 16 wherein the LNP formulation comprises a combined molar percentage of DLin-MC3-DMA and at least one helper lipid in a range from about 20 w / w% to about 80 w / w% 19. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 16 wherein the LNP formulation comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 5 to about 300. 53 170450739.

120. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 19 wherein the LNP formulation comprises a weight ratio of cholesterol to DMG-PEG2000, ranging from about 10 to about 200.

21. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 20 wherein the LNP formulation comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 0.1 to 200.

22. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 20 wherein the LNP formulation comprises a weight ratio of DLin-MC3-DMA to the helper lipid, from 1 to 100.

23. The lipid nanoparticle (LNP) formulation of any one of claims 1 to 22 wherein the LNP formulation comprises one or more gene editing agents, carbohydrates, protein, lipids, nucleic acids or combinations thereof.

24. A method of intracellular delivery of a therapeutic agent comprising, contacting a cell or administering to a subject a lipid nanoparticle (LNP) comprising an effective amount of a therapeutic agent.

25. A method of intracellular delivery of a therapeutic agent comprising, contacting a cell or administering to a subject a lipid nanoparticle formulation of any one of claims 1 to 23.

26. The method of claim 24, wherein the lipid nanoparticle (LNP) or lipid nanoparticle formulation comprises: an ionizable lipid, at least one helper lipid, polyethylene glycol (PEG)- modified lipid, a sterol or combinations thereof.

27. The method of claim 26, wherein the at least one helper lipid comprises a cationic lipid, a zwitterionic lipid, an anionic lipid, or combinations thereof.

28. The method of claim 27 wherein the cationic lipid comprises 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) or the combination thereof.

29. The method of claim 27 or 28 wherein the zwitterionic lipid comprises 1,2-dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), distearoylphosphatidylcholine (DSPC) or the combination thereof. 54 170450739.

130. The method of cany one of claims 27 to 29 wherein the anionic lipid comprises 1,2- dimyristoyl-sn-glycero-3-phosphate (14PA), 1-stearoyl-2-oleoyl-sn-glycero-3-phospho (1’-rac- glycerol) (18PG) or the combination thereof.

31. The method of any one of claims 24 to 30 wherein the ionizable lipid comprises DLin- MC3-DMA.

32. The method of any one of claims 24 to 31 wherein the polyethylene glycol (PEG)- modified lipid comprises DMG-PEG 2000.

33. The method of any one of claims 24 to 32 wherein the sterol comprises cholesterol.

34. The method of c any one of claims 24 to 33 wherein the therapeutic agent is encapsulated by the LNP.

35. The method of any one of claims 24 to 34 wherein the lipid nanoparticle (LNP) or the lipid nanoparticle formulation comprises one or more gene editing agents, carbohydrates, protein, lipids, nucleic acids or combinations thereof.

36. A method of treating a subject, comprising administering to the subject a lipid nanoparticle formulation of any one of claims 1-23, and treating the subject.

37. The method of claim 36, wherein the a lipid nanoparticle formulation comprises one or more therapeutic agents.

38. The method of claim 37 wherein the one or more therapeutic agents comprise carbohydrate, protein, lipids, nucleic acids or combinations thereof.

39. The method of claim 36 or 37 wherein the therapeutic agent is a gene editing complex comprising a CRISPR / Cas system.

40. A lipid nanoparticle (LNP) formulation comprising an ionizable lipid, at least one helper lipid, a polyethylene glycol (PEG)-modified lipid, a sterol or combinations thereof.

41. The lipid nanoparticle formulation (LNP) of claim 40, wherein the LNP comprises an ionizable lipid in a range from about 20% to about 90%; a helper lipid in a range from about 0.1% to about 60%; a polyethylene glycol (PEG)-modified lipid in a range from about 0.1% to about 5%; a sterol in a range from about 10% to about 60% by weight. 55 170450739.1

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