Compositions and methods for delivery of nucleic acids

Abstract

The present disclosure provides compositions and methods for genetic modification of cells, including but not limited to hepatocytes. The compositions and methods may include lipid nanoparticles, which include at least one bioreducible ionizable cationic lipid, at least one structured lipid, at least one phospholipid, and at least one PEGylated lipid.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 152,517, filed February 23, 2021, U.S. Provisional Patent Application No. 63 / 156,649, filed March 4, 2021, U.S. Provisional Patent Application No. 63 / 164,174, filed March 22, 2021, and U.S. Provisional Patent Application No. 63 / 197,946, filed June 7, 2021. The contents of each of the above-mentioned patent applications are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on February 23, 2022 is named "POTH-066_001WO_SeqList.txt" and is approximately 320,338 bytes in size.

[0003] The present disclosure relates generally to novel lipid nanoparticles (LNPs) comprising bioreducible, ionizable cationic lipids, methods for preparing these LNPs, and the use of these LNPs for gene therapy and cell-based therapy applications. [Background technology]

[0004] There is a long-felt and unmet need in the art for compositions and methods for delivering nucleic acids to cells and for genetically modifying cells in vivo, ex vivo, and in vitro. Widely accepted gene delivery and genetic modification techniques, such as the use of viral vectors, including AAV, can cause acute toxicity and adverse side effects in patients. The present disclosure provides improved compositions, methods, and kits for the delivery of nucleic acids to various types of cells, including hepatocytes, in vivo, ex vivo, and in vitro. More specifically, the present disclosure provides improved lipid nanoparticle compositions and methods for using the same. These lipid nanoparticle compositions and methods allow for the delivery of certain types of nucleic acids (e.g., RNA) to liver cells with high efficiency and low toxicity. Furthermore, the lipid nanoparticle compositions of the present disclosure exhibit improved storage stability, which is advantageous in clinical and commercial settings. Thus, the compositions and methods of the present disclosure have broad applicability to a variety of fields, including gene therapy and the production of cell-based therapeutics. Summary of the Invention

[0005] In some embodiments, novel lipid nanoparticles (LNPs) are provided that include a bioreducible, ionizable cationic lipid. In one embodiment, the bioreducible, ionizable cationic lipid is Coatsome SS-OP.

[0006] In one aspect, a pharmaceutical composition is provided comprising a composition of the present disclosure and at least one pharma- ceutically acceptable excipient or diluent.

[0007] In one aspect, a method of delivering at least one nucleic acid to at least one cell is provided, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0008] In one aspect, a method of genetically modifying at least one cell is provided, comprising contacting at least one cell with at least one composition of the present disclosure.

[0009] In one aspect, a method of treating at least one disease or disorder in a subject in need thereof is provided, comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure.

[0010] In one aspect, a method of delivering at least one nucleic acid to at least one cell is provided, comprising contacting the at least one cell with at least one composition of the present disclosure.

[0011] In one aspect, a cell modified according to the methods of the disclosure is provided.

[0012] Any of the above aspects may be combined with any other aspect.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification, the singular also includes the plural unless the context clearly indicates otherwise, and by way of example, the terms "a", "an" and "the" are understood to be singular or plural, and the term "or" is understood to be inclusive. As an example, an "element" means one or more elements. Throughout this specification, the word "comprising" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless the context clearly indicates otherwise, all numerical values ​​provided herein are modified by the term "about."

[0014] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. References cited herein are not admitted to be prior art to the claimed invention. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims. [Brief description of the drawings]

[0015] The above and further features will be more clearly understood from the following detailed description when considered in conjunction with the accompanying drawings.

[0016] [Figure 1] 1 is a series of graphs showing human FVIII expression in adult mice administered LNPs of the present disclosure. [Diagram 2] 1 is a series of graphs showing expression of human FIX in mice administered LNPs of the present disclosure. [Diagram 3] 1 is a series of graphs showing the mean diameter and polydispersity index (PDI) of various LNP compositions of the present disclosure over the course of a 7-day incubation at 0.1 mg / mL and a temperature of 4° C. [Figure 4] 1 is a graph showing human FVIII expression in mice administered LNPs of the present disclosure. [Diagram 5] 1 is a graph showing expression of human erythropoietin (hEPO) in non-human primates (NHPs) administered LNPs of the present disclosure. [Figure 6] 1 is a series of graphs showing liver enzyme levels in NHPs administered LNPs of the present disclosure. [Figure 7]1 is a graph showing survival of mice administered human OTC (hOTC) transposon AAV viral vector particles and human OTC (hOTC) transposon AAV viral vector particles in combination with SPB LNPs of the present disclosure. [Figure 8] 1 is a series of graphs showing integrated viral copy number (VCN) and hOTC mRNA levels in mice administered human OTC (hOTC) transposon AAV viral vector particles in combination with SPB LNPs of the present disclosure. [Figure 9] 1 is a series of graphs showing survival and orotic acid levels in mice administered human OTC (hOTC) transposon AAV viral vector particles in combination with SPB LNPs of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present disclosure provides novel lipid nanoparticle (LNP) compositions comprising bioreducible ionizable cationic lipids, methods for preparing the compositions, and methods for using the compositions. In a non-limiting example, the compositions and methods of the present disclosure can be used for gene delivery in the context of gene therapy. In another non-limiting example, the compositions and methods of the present disclosure can be used in the context of cell-based therapeutics. In another non-limiting example, the compositions and methods of the present disclosure can be used broadly to deliver nucleic acids that induce expression of therapeutic proteins, including, but not limited to, secreted therapeutic proteins. In a non-limiting example, the compositions and methods of the present disclosure can be used broadly to deliver nucleic acids to liver cells in vivo, ex vivo, or in vitro for the treatment of certain liver disorders.

[0018] The bioreducible, ionizable cationic lipids of the present disclosure are biodegradable, thereby allowing them to be broken down and metabolized in animals. Without intending to be bound by theory, this bioreducibility advantageously reduces cationic lipid-associated cytotoxicity.

[0019] In some embodiments of the compositions and methods of the present disclosure, the bioreducible, ionizable cationic lipid for use in the LNP composition can be ssPalmO-Ph-P4C2. As will be appreciated by those of skill in the art, ssPalmO-Ph-P4C2 has the following structure: [ka]

[0020] As will be appreciated by those skilled in the art, ssPalmO-Ph-P4C2 may also be referred to as Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph. Thus, ssPalmO-Ph-P4C2, Coatsome® SS-OP, ssPalmO-Phe-P4C2, ssPalmO-Phenyl-P4C2, ssPalmO-Phe, and ssPalmO-Ph are used interchangeably herein to refer to the bioreducible, ionizable cationic lipid having the chemical structure represented by Formula I.

[0021] Without intending to be bound by theory, three specific segments of ssPalmO-Ph-P4C2 facilitate its biodegradation. First, the tertiary amine of each piperzine ring is an acidic pH-responsive cationic charged unit. Upon endocytosis, the tertiary amine moieties become positively charged in response to the acidic intracellular endosomal compartment. Here, they can interact with and destabilize membranes, which leads to endosomal escape. Once in the cytosol, the disulfide bond is susceptible to reduction by glutathione, generating two free sulfhydryl groups. The resulting high concentration of free thiols further leads to nucleophilic reactions, and the particle autolyzes / disintegrates via thioesterification, releasing the payload into the cytosol. This is defined as Hyper or hydrolysis accelerated by intraparticle concentration of reactants, potentially eliminating the potential toxic side effects of cationic lipids in general.

[0022] Compositions of the Disclosure - Lipid Nanoparticles The present disclosure provides a composition comprising at least one lipid nanoparticle, comprising at least one cationic lipid and at least one nucleic acid molecule.In some embodiments, the lipid nanoparticle can further comprise at least one structured lipid.In some embodiments, the lipid nanoparticle can further comprise at least one phospholipid.In some embodiments, the lipid nanoparticle can further comprise at least one PEGylated lipid.

[0023] Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, the at least one lipid nanoparticle comprising at least one cationic lipid, at least one nucleic acid molecule, at least one structured lipid, at least one phospholipid, and at least one PEGylated lipid.

[0024] Bioreducible ionizable cationic lipids In some embodiments, the cationic lipid can be a bioreducible ionizable cationic lipid.Thus, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises at least one bioreducible ionizable cationic lipid.

[0025] As used herein, the term "bioreducible ionizable cationic lipid" is used in its broadest sense to refer to a cationic lipid that contains at least one tertiary amine, at least one disulfide group, a bond that is susceptible to cleavage by thioesterification, and at least one group that further contains at least two saturated or unsaturated hydrocarbon chains. Exemplary bioreducible ionizable cationic lipids include, but are not limited to, those described in Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the contents of which are incorporated herein by reference in their entirety.

[0026] In some embodiments, the bioreducible ionizable cationic lipid can comprise at least two tertiary amines. In some embodiments, at least one tertiary amine can be a substituted piperidinyl group. In some embodiments, each tertiary amine can be a substituted piperidinyl group. In some embodiments, the bioreducible ionizable cationic lipid can comprise at least one disulfide bond. In some embodiments, the sulfur atom of the disulfide bond is bonded to the nitrogen of the piperidinyl ring through an alkylene group, thereby forming two tertiary amine groups adjacent to the disulfide bond. In some embodiments, at least one of the alkylene groups is an ethylene group. In some embodiments, each of the alkylene groups is an ethylene group.

[0027] In some embodiments, at least one group that comprises a bond that is susceptible to cleavage by thioesterification can be a phenyl ester group. In some embodiments, the bioreducible ionizable cationic lipid can comprise at least two phenyl ester groups. In some embodiments, at least one of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some embodiments, each of the at least two saturated or unsaturated hydrocarbon chains is an unsaturated hydrocarbon chain. In some embodiments, the unsaturated hydrocarbon chain can be octadecene. In some embodiments, the octadecene can be (Z)-octadec-9-ene. In some embodiments, the (Z)-octadec-9-ene group can be bonded to the phenyl ester group of the bioreducible ionizable cationic lipid.

[0028] Exemplary bioreducible ionizable cationic lipids and methods for preparing such lipids useful in the methods of the present disclosure include those disclosed in International Patent Application No. PCT / JP2016 / 052690, published as WO / 2016 / 121942, and International Patent Application No. PCT / JP2019 / 012302, published as WO / 2019 / 188867, the contents of each of which are incorporated herein by reference in their entirety. Thus, the present disclosure provides a composition comprising at least one lipid nanoparticle, wherein the at least one lipid nanoparticle comprises any one of the bioreducible ionizable cationic lipids described in WO / 2016 / 121942 and WO / 2019 / 188867.

[0029] Accordingly, the present disclosure provides a composition comprising at least one lipid nanoparticle, the at least one lipid nanoparticle comprising at least one bioreducible ionizable cationic lipid, at least one nucleic acid molecule, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid.

[0030] In some embodiments, the bioreducible, ionizable cationic lipid can be ssPalmO-Ph-P4C2 having a structure represented by Formula I (see Akita et al., (2020) Biol. Phar. Bull. 43:1617-1625, the contents of which are incorporated by reference in their entirety).

[0031] As described herein, the LNP compositions of the present disclosure that comprise at least one bioreducible ionizable cationic lipid advantageously exhibit significantly reduced toxicity in animals compared to LNP compositions that comprise non-bioreducible ionizable cationic lipid.In particular, administration of the LNP compositions of the present disclosure surprisingly does not result in any weight loss.In fact, the LNP compositions of the present disclosure are so non-toxic that animals administered the LNPs actually gain weight, even when an amount of LNP that exceeds the lethal dose of the LNP compositions that comprise non-bioreducible ionizable cationic lipid is administered.

[0032] LNP component In some embodiments, the LNPs of the disclosure may comprise about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one bioreducible ionizable cationic lipid.

[0033] In some embodiments, the LNPs of the disclosure are at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%, or at least The lipid composition may comprise about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one bioreducible ionizable cationic lipid.

[0034] In some embodiments, the LNPs of the disclosure may comprise about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one structured lipid.

[0035] In some embodiments, the LNPs of the disclosure are at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%. %, or at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one structured lipid.

[0036] In some embodiments, the LNPs of the disclosure may comprise about 2.5 mol%, or about 5 mol%, or about 7.5 mol%, or about 10 mol%, or about 12.5 mol%, or about 15 mol%, or about 17.5 mol%, or about 20 mol%, or about 22.5 mol%, or about 25 mol%, or about 27.5 mol%, or about 30 mol%, or about 32.5 mol%, or about 35 mol%, or about 37.5 mol%, or about 40 mol%, or about 42.5 mol%, or about 45 mol%, or about 47.5 mol%, or about 50 mol%, or about 52.5 mol%, or about 55 mol%, or about 57.5 mol%, or about 60 mol%, or about 62.5 mol%, or about 65 mol%, or about 67.5 mol%, or about 70 mol% of at least one phospholipid.

[0037] In some embodiments, the LNPs of the disclosure are at least about 2.5 mol%, or at least about 5 mol%, or at least about 7.5 mol%, or at least about 10 mol%, or at least about 12.5 mol%, or at least about 15 mol%, or at least about 17.5 mol%, or at least about 20 mol%, or at least about 22.5 mol%, or at least about 25 mol%, or at least about 27.5 mol%, or at least about 30 mol%, or at least about 32.5 mol%, or at least about 35 mol%. %, or at least about 37.5 mol%, or at least about 40 mol%, or at least about 42.5 mol%, or at least about 45 mol%, or at least about 47.5 mol%, or at least about 50 mol%, or at least about 52.5 mol%, or at least about 55 mol%, or at least about 57.5 mol%, or at least about 60 mol%, or at least about 62.5 mol%, or about 65 mol%, or at least about 67.5 mol%, or at least about 70 mol% of at least one phospholipid.

[0038] In some embodiments, the LNPs of the disclosure may comprise about 0.25 mol%, or about 0.5 mol%, or about 0.75 mol%, or about 1.0 mol%, or about 1.25 mol%, or about 1.5 mol%, or about 1.75 mol%, or about 2.0 mol%, or at least about or about 2.5 mol%, or about 5 mol% of at least one PEGylated lipid.

[0039] In some embodiments, the LNPs of the disclosure may comprise at least about 0.25 mol%, or at least about 0.5 mol%, or at least about 0.75 mol%, or at least about 1.0 mol%, or at least about 1.25 mol%, or at least about 1.5 mol%, or at least about 1.75 mol%, or at least about 2.0 mol%, or at least about or at least about 2.5 mol%, or at least about 5 mol% of at least one PEGylated lipid.

[0040] structural lipids In some aspects, the structured lipids may be steroids. In some aspects, the structured lipids may be sterols. In some aspects, the structured lipids may comprise cholesterol. In some aspects, the structured lipids may comprise ergosterol. In some aspects, the structured lipids may be phytosterols.

[0041] Phospholipids As used herein, the term "phospholipid" is used in the broadest sense to refer to any amphipathic molecule that contains a polar (hydrophilic) head group that contains phosphate and two hydrophobic fatty acid chains.

[0042] In some aspects of the lipid nanoparticles of the present disclosure, the phospholipid may include dioleoylphosphatidylethanolamine (DOPE).

[0043] In some embodiments of the lipid nanoparticles of the present disclosure, the phospholipid may include DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0044] In some embodiments of the lipid nanoparticles of the present disclosure, the phospholipid may include DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine).

[0045] In some embodiments, the phospholipid is selected from the group consisting of DDPC (1,2-didecanoyl-sn-glycero-3-phosphocholine), DEPA-NA (1,2-dierucoyl-sn-glycero-3-phosphate (sodium salt)), DEPC (1,2-dierucoyl-sn-glycero-3-phosphocholine), DEPE (1,2-dierucoyl-sn-glycero-3-phosphoethanolamine), DEPG-NA (1,2-dierucoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DLOPC (1,2-dilinoleoyl-sn -glycero-3-phosphocholine), DLPA-NA (1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt)), DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DLPG-NA (1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DLPG-NH4 (1,2-dilauroyl-sn-glycero-3[phospho-rac-(1-glycerol) (ammonium salt)), (sodium salt)), DLPS-NA (1,2-dilauroyl-sn-glycero-3-phosphoserine (sodium salt)), DMPA-NA (1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt)), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DMPG-NA (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), DMPG-NH4 (1,2-dimyristoyl-sn-glycero-3[phospho-rac-(1-glycerol) (sodium salt)), 1,2-dioleoyl-sn-glycero-3-phosphoserine (sodium salt)), DMPG-NH4 / NA (1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DMPS-NA (1,2-dimyristoyl-sn-glycero-3-phosphoserine (sodium salt)), DOPA-NA (1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt)), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPG-NA (1,2-dioleoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DOPS-NA (1,2-dioleoyl-sn-glycero-3-phosphoserine(sodium salt)), DPPA-NA (1,2-dipalmitoyl-sn-glycero-3-phosphate(sodium salt)), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DPPG-NA (1,2-dipalmitoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DPPG-NH4 (1,2-dipalmitoyl- sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DPPS-NA (1,2-dipalmitoyl-sn-glycero-3-phosphoserine (sodium salt)), DSPA-NA (1,2-distearoyl-sn-glycero-3-phosphate (sodium salt)), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPG-NA (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(sodium salt)), DSPG-NH4 (1,2-distearoyl-sn-glycero-3[phospho-rac-(1-glycerol)(ammonium salt)), DSPS-NA(1,2-distearoyl-sn-glycero-3-phosphoserine (sodium salt)), EPC (Egg-PC), HEPC (Hydrogenated Egg PC), HSPC (Hydrogenated Soy PC), LYSOPC MYRISTIC (1-Myristoyl-sn-glycero-3-phosphocholine), LYSOPC PALMITIC (1-Palmitoyl-sn-glycero-3-phosphocholine), LYSOPC STEARIC (1-stearoyl-sn-glycero-3-phosphocholine), milk sphingomyelin (MPPC, 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine), MSPC (1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine), PMPC (1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanol). amine), POPG-NA (1-palmitoyl-2-oleoyl-sn-glycero-3[phospho-rac-(1-glycerol)](sodium salt)), PSPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine), SMPC (1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine), SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine), SPPC (1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine), or any combination thereof.

[0046] PEGylated lipids As used herein, the term "PEGylated lipid" is used to refer to any lipid that is modified (for example) with (covalently attached to) at least one polyethylene glycol molecule. In some embodiments, the PEGylated lipid may comprise 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, hereinafter referred to as DMG-PEG2000.

[0047] nucleic acid In some embodiments, the lipid nanoparticles may contain at least one nucleic acid molecule. In some embodiments, the lipid nanoparticles may contain multiple nucleic acid molecules. In some embodiments, at least one nucleic acid molecule or multiple nucleic acid molecules may be formulated in the lipid nanoparticles.

[0048] In some embodiments, the nucleic acid molecule can be a synthetic nucleic acid molecule. In some embodiments, the nucleic acid molecule can be a non-naturally occurring nucleic acid molecule. In some embodiments, the non-naturally occurring nucleic acid molecule can comprise at least one non-naturally occurring nucleotide. The at least one non-naturally occurring nucleotide can be any non-naturally occurring nucleotide known in the art. In some embodiments, the nucleic acid molecule can be a modified nucleic acid molecule. In some embodiments, the modified nucleic acid molecule can comprise at least one modified nucleotide. The at least one modified nucleotide can be any modified nucleic acid known in the art.

[0049] In some aspects, the lipid nanoparticles may comprise lipids and nucleic acids in a specified ratio (weight / weight).

[0050] In some embodiments, lipid nanoparticles comprising at least one nucleic acid molecule have a lipid to nucleic acid ratio of about 5:1 to about 15:1, or about 10:1 to about 20:1, or about 15:1 to about 25:1, or about 20:1 to about 30:1, or about 25:1 to about 35:1, or about 30:1 to about 40:1, or about 35:1 to about 45:1, or about 40:1 to about 50:1, or about 45:1 to about 55:1, or about 50:1 to about 60:1, or about 55:1 to about 65:1, or about 60:1 to about 70:1, or about 65:1 to about 75:1, or about 70:1 to about 80:1, or in a lipid:nucleic acid weight / weight ratio of about 75:1 to about 85:1, or about 80:1 to about 90:1, or about 85:1 to about 95:1, or about 90:1 to about 100:1, or about 95:1 to about 105:1, or about 100:1 to about 110:1, or about 105:1 to about 115:1, or about 110:1 to about 120:1, or about 115:1 to about 125:1, or about 120:1 to about 130:1, or about 125:1 to about 135:1, or about 130:1 to about 140:1, or about 135:1 to about 145:1, or about 140:1 to about 150:1.

[0051] In some embodiments, the lipid nanoparticles have a lipid to nucleic acid ratio of about 5:1, or about 10:1, or about 15:1, or about 20:1, or about 25:1, or about 30:1, or about 35:1, or about 40:1, or about 45:1, or about 50:1, or about 55:1, or about 60:1, or about 65:1, or about 70:1, or about 75:1, or about 80:1 or about 85:1, or about 90:1, or about 95:1, or about 100:1, or about 105:1, or about 110:1, or about 115:1, or about 120:1, or about 125:1, or about 130:1, or about 135:1, or about 140:1, or about 145:1, or about 150:1, or about 200:1 lipid:nucleic acid weight / weight ratio.

[0052] In some embodiments, the lipid nanoparticles may comprise lipid and nucleic acid in a lipid:nucleic acid weight / weight ratio of about 10:1, or about 17.5:1, or about 25:1.

[0053] In some embodiments, the nucleic acid molecule can be an RNA molecule.Thus, in some embodiments, the lipid nanoparticle can comprise at least one RNA molecule.In some embodiments, the RNA molecule can be an mRNA molecule.In some embodiments, the mRNA molecule can comprise 5'-CAP.

[0054] In some embodiments, the mRNA molecule may be capped using any method and / or capping moiety known in the art. The mRNA molecule may be capped with a m7G(5')ppp(5')G moiety. The m7G(5')ppp(5')G moiety is also referred to herein as "Cap0". The mRNA molecule may be capped with a CleanCap® moiety. The CleanCap® moiety may comprise a m7G(5')ppp(5')(2'OMeA) (CleanCap® AG) moiety. The CleanCap® moiety may comprise a m7G(5')ppp(5')(2'OMeG) (CleanCap® GG) moiety. The mRNA molecule may be capped with an anti-reverse cap analog (ARCA®) moiety. The ARCA® moiety may comprise a m7(3'-O-methyl)G(5')ppp(5')G moiety. The mRNA molecule can be capped with a CleanCap® 3'OMe moiety (CleanCap®+ARCA®).

[0055] In some embodiments, the mRNA molecule may contain at least one modified nucleic acid.

[0056] Modified nucleic acids include, but are not limited to, 5-methoxyuridine (5moU), N1-methylpseudouridine (me 1 Ψ), pseudouridine (Ψ), and 5-methylcytidine (5-MeC).

[0057] In some embodiments, the nucleic acid molecule can be a DNA molecule. Thus, in some embodiments, the lipid nanoparticle can include at least one DNA molecule. In some embodiments, the DNA molecule can be a circular DNA molecule, such as, but not limited to, a DNA plasmid. In some embodiments, the lipid nanoparticle can include a DNA plasmid. In some embodiments, the DNA molecule can be a linear DNA molecule, such as, but not limited to, a linear DNA plasmid. In some embodiments, the DNA molecule can be a DoggyBone DNA molecule. In some embodiments, the DNA molecule can be a DNA nanoplasmid.

[0058] The DNA plasmid may be at least about 0.25 kb, or at least about 0.5 kb, or at least about 0.75 kb, or at least about 1.0 kb, or at least about 1.25 kb, or at least about 1.5 kb, or at least about 1.75 kb, or at least about 2.0 kb, or at least about 2.25 kb, or at least about 2.5 kb, or at least about 2.75 kb, or at least about 3.0 kb, or at least about 3.25 kb, or at least about 3.5 kb, or at least about 3.75 kb, or at least about 4.0 kb, or at least about 4.25 kb, or at least about 4.5 kb, or at least about 4.75 kb, or at least about 5.0 kb, or at least about 5.25 kb, or at least about 5.5 kb, or at least about 5.75 kb, or at least about 6.0 kb, or at least about 6.25 kb, or at least about 6.5 kb, or at least about 6.75 kb, or at least about 7.0 kb, or at least about 7.25 kb, or at least about 7.5 kb, or at least about 7.75 kb b, or at least about 8.0 kb, or at least about 8.25 kb, or at least about 8.5 kb, or at least about 8.75 kb, or at least about 9.0 kb, or at least about 9.25 kb, or at least about 9.5 kb, or at least about 9.75 kb, or at least about 10.0 kb, or at least about 10.25 kb, or at least about 10.5 kb, or at least about 10.75 kb, or at least about 11.0 kb, or at least about 11.25 kb, or at least about 11.5 kb, or may include those which may be at least about 11.75 kb, or at least about 12 kb, or at least about 12.25 kb, or at least about 12.5 kb, or at least about 12.75 kb, or at least about 13.0 kb, or at least about 13.25 kb, or at least about 13.5 kb, or at least about 13.75 kb, or at least about 14.0 kb, or at least about 14.25 kb, or at least about 14.5 kb, or at least about 14.75 kb, or at least about 15.0 kb in length.

[0059] LNP composition In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one structured lipid.In some embodiments, the lipid nanoparticles can comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, and at least one PEGylated lipid.In some embodiments, the at least one bioreducible ionizable cationic lipid can be ssPalmO-Ph-P4C2.

[0060] In some embodiments, the at least one structured lipid can be a mixture of two structured lipids. In some embodiments, the at least one PEGylated lipid can be a mixture of two PEGylated lipids.

[0061] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structured lipid, at least one PEGylated lipid, or any combination thereof. In some embodiments, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0062] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, and at least one PEGylated lipid. In some embodiments, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0063] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structured lipid, at least one phospholipid, at least one PEGylated lipid, or any combination thereof. In some embodiments, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0064] In some embodiments, the lipid nanoparticles may comprise at least one nucleic acid molecule, at least one bioreducible ionizable cationic lipid, at least one structural lipid, at least one phospholipid, and at least one PEGylated lipid. In some embodiments, the at least one bioreducible ionizable cationic lipid may be ssPalmO-Ph-P4C2.

[0065] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 28 mol% ssPalmO-Ph-P4C2, about 60 mol% cholesterol, about 10 mol% DOPE, and about 2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 10:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0066] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 29.5 mol% cholesterol, about 10 mol% DOPE, and about 0.5 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0067] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 32.92 mol% ssPalmO-Ph-P4C2, about 32.92 mol% cholesterol, about 32.92 mol% DOPE, and about 1.25 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 55:1 (w / w).

[0068] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 10 mol% ssPalmO-Ph-P4C2, about 29.5 mol% cholesterol, about 60 mol% DOPE, and about 0.5 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0069] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 28 mol%-29 mol% cholesterol, about 10 mol% DOPE, and about 1.25 mol%-2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0070] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 28 mol% cholesterol, about 10 mol% DOPE, and about 2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0071] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 28.75 mol% cholesterol, about 10 mol% DOPE, and about 1.25 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0072] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0073] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 27.84 mol% ssPalmO-Ph-P4C2, about 56.25 mol% cholesterol, about 13.46 mol% DOPE, and about 2.45 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 88:1 (w / w).

[0074] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 27.9 mol% ssPalmO-Ph-P4C2, about 51.51 mol% cholesterol, about 18.59 mol% DOPE, and about 2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0075] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 26.4 mol% cholesterol, about 11.6 mol% DOPE, and about 2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 70:1 (w / w).

[0076] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 30.37 mol% ssPalmO-Ph-P4C2, about 37.27 mol% cholesterol, about 30.36 mol% DOPE, and about 2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0077] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% to 59 mol% ssPalmO-Ph-P4C2, about 30 mol% to 40 mol% cholesterol, about 5 mol% to 10 mol% DOPC, DSPC, or DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 to about 100:1 (w / w).

[0078] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0079] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0080] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0081] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0082] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0083] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0084] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0085] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0086] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0087] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0088] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0089] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0090] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0091] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0092] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0093] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0094] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0095] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0096] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0097] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0098] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0099] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0100] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0101] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0102] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0103] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0104] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0105] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0106] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0107] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0108] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0109] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0110] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0111] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 75:1 (w / w).

[0112] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 100:1 (w / w).

[0113] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0114] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 49 mol% to 60 mol% ssPalmO-Ph-P4C2, about 32 mol% to 44 mol% cholesterol, about 5 mol% DOPC, and about 1.5 mol% to 3.0 mol% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 to about 100:1 (w / w).

[0115] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% to 59 mol% ssPalmO-Ph-P4C2, about 30 mol% to 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 to about 100:1 (w / w).

[0116] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 to about 100:1 (w / w).

[0117] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 to about 100:1 (w / w).

[0118] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, wherein the at least one lipid nanoparticle comprises at least one nucleic acid molecule, wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and wherein the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 to about 100:1 (w / w).

[0119] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 49.4 mol% ssPalmO-Ph-P4C2, about 44 mol% cholesterol, about 5 mol% DOPC, and about 1.6 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 60:1 (w / w).

[0120] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 32.8 mol% cholesterol, about 5 mol% DSPC, and about 2.2 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 60:1 (w / w).

[0121] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 34 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 40:1 (w / w).

[0122] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 41.8 mol% ssPalmO-Ph-P4C2, about 52.2 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one mRNA molecule. In some embodiments, the mRNA molecule further comprises 5'-CAP. In some embodiments, the ratio of lipid to nucleic acid in at least one nanoparticle may be about 60:1 (w / w).

[0123] In some embodiments, the nucleic acid molecule is a DNA molecule. Thus, the present disclosure provides lipid nanoparticles comprising at least one nucleic acid molecule, which may comprise about 22.71 mol% ssPalmO-Ph-P4C2, about 55.21 mol% cholesterol, about 20.89 mol% DOPE, and about 1.25 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA (see WO / 2020 / 154645). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 100:1 (w / w).

[0124] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 50 mol% ssPalmO-Ph-P4C2, about 38.6 mol% cholesterol, about 10 mol% DOPE, and about 1.4 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA (see WO / 2020 / 154645). In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 200:1 (w / w).

[0125] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 34.69 mol% ssPalmO-Ph-P4C2, about 39.74 mol% cholesterol, about 24.69 mol% DOPE, and about 1.25 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 50:1 (w / w).

[0126] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 50 mol% ssPalmO-Ph-P4C2, about 20 mol% cholesterol, about 29.5 mol% DOPE, and about 0.5 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 100:1 (w / w).

[0127] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 50 mol% ssPalmO-Ph-P4C2, about 20 mol% cholesterol, about 28.7 mol% DOPE, and about 1.3 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 50:1 (w / w).

[0128] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% to 59 mol% ssPalmO-Ph-P4C2, about 30 mol% to 40 mol% cholesterol, about 5 mol% to 10 mol% DOPC, DSPC, or DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 to about 100:1 (w / w).

[0129] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0130] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0131] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0132] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0133] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0134] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0135] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0136] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0137] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0138] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0139] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0140] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0141] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0142] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0143] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0144] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0145] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0146] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0147] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0148] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0149] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0150] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0151] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0152] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DSPC, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0153] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0154] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0155] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0156] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 32.5 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0157] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0158] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 30 mol% cholesterol, about 10 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0159] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0160] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0161] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0162] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0163] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 100:1 (w / w).

[0164] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% DOPE, and about 1 mol% DMG-PEG2000, and the lipid nanoparticles further comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule may be a DNA nanoplasmid. In some embodiments, the at least one DNA molecule may be a covalently closed end DNA. In some embodiments, the ratio of lipid to nucleic acid in the at least one nanoparticle may be about 75:1 (w / w).

[0165] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 48 mol% to about 61 mol% ssPalmO-Ph-P4C2, about 31 mol% to about 53 mol% cholesterol, about 4 mol% to about 11 mol% phospholipid, and about 0.5 mol% to about 3 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipids to nucleic acids in the nanoparticles can be about 30:1 to about 110:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 40:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 60:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 100:1 (w / w).

[0166] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 41.8 mol% to about 60 mol% ssPalmO-Ph-P4C2, about 32.8 mol% to about 52.2 mol% cholesterol, about 5 mol% to about 10 mol% phospholipid, and about 1 mol% to about 2.2 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipids to nucleic acids in the nanoparticles can be about 30:1 to about 110:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 40:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 60:1 (w / w). In some embodiments, the ratio of lipids to nucleic acids can be about 100:1 (w / w).

[0167] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0168] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 49.4 mol% ssPalmO-Ph-P4C2, about 44 mol% cholesterol, about 5 mol% phospholipid, and about 1.6 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0169] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 32.8 mol% cholesterol, about 5 mol% phospholipid, and about 2.2 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0170] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 60 mol% ssPalmO-Ph-P4C2, about 34 mol% cholesterol, about 5 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0171] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 41.8 mol% ssPalmO-Ph-P4C2, about 52.2 mol% cholesterol, about 5 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0172] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 30:1 to about 110:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 40:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 60:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0173] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 53 mol% to about 60 mol% ssPalmO-Ph-P4C2, about 34 mol% to about 41 mol% cholesterol, about 4 mol% to about 11 mol% phospholipid, and about 0.5 mol% to about 2 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 70:1 to about 105:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid can be about 75:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid can be about 100:1 (w / w).

[0174] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% to about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% to about 40 mol% cholesterol, about 5 mol% to about 10 mol% phospholipid, and about 0.5 mol% to about 1.5 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticles can be about 70:1 to about 105:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid can be about 75:1 (w / w). In some embodiments, the ratio of lipid to nucleic acid can be about 100:1 (w / w).

[0175] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 10 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 75:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0176] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 59 mol% ssPalmO-Ph-P4C2, about 35 mol% cholesterol, about 5 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 75:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0177] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 54 mol% ssPalmO-Ph-P4C2, about 40 mol% cholesterol, about 5 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 75:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0178] In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 56.5 mol% ssPalmO-Ph-P4C2, about 37.5 mol% cholesterol, about 5 mol% phospholipid, and about 1 mol% DMG-PEG2000. In some embodiments, the at least one nucleic acid molecule comprises at least one RNA molecule. In some embodiments, the at least one RNA molecule may be mRNA. In some embodiments, the mRNA molecule may further comprise 5'-CAP. In some embodiments, the at least one nucleic acid molecule may comprise at least one DNA molecule. In some embodiments, the at least one DNA molecule may be DoggyBone DNA or DNA nanoplasmid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the phospholipid may be DOPC. In some embodiments, the phospholipid may be DSPC. In some embodiments, the ratio of lipid to nucleic acid in the nanoparticle may be about 70:1 to about 105:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 75:1 (w / w). In some embodiments, the lipid to nucleic acid ratio can be about 100:1 (w / w).

[0179] In some embodiments of the present disclosure, the lipid nanoparticle or nanoparticles may be stable for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days at about 2° C. to about 6° C., or about 4° C. In some embodiments of the present disclosure, the lipid nanoparticles may be stable at about 4° C. for at least about 7 days.

[0180] In some embodiments of the present disclosure, the lipid nanoparticle or nanoparticles may be stable for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, or about 14 days at about 2° C. to about 6° C., or about 4° C. In some embodiments of the present disclosure, the lipid nanoparticle or nanoparticles may be stable for about 7 days at about 4° C.

[0181] In some embodiments, a lipid nanoparticle, or a plurality of lipid nanoparticles, can be said to be stable if there is a change in diameter (in the case of a single lipid nanoparticle) or average diameter (in the case of a plurality of lipid nanoparticles) of about 0.5% or less, about 1% or less, about 5% or less, about 10% or less, about 15% or less, about 20% or less, about 25% or less, or about 30% or less, about 35% or less, about 40% or less, about 45% or less, or about 50% or less. The diameter of a lipid nanoparticle or the average diameter of a plurality of nanoparticles can be determined using any standard method known in the art, as will be appreciated by those of skill in the art.

[0182] In some embodiments, lipid nanoparticles can be said to be stable when there is a change in polydispersity index (PDI) or lipid nanoparticles of about 0.5% or less, about 1% or less, or about 5% or less, or about 10% or less, or about 15% or less, or about 20% or less, or about 25% or less, or about 30% or less, or about 35% or less, or about 40% or less, or about 45% or less, or about 50% or less. The PDI of lipid nanoparticles can be determined using any standard method known in the art, as will be understood by those skilled in the art.

[0183] C12-200LNP of the present disclosure The present disclosure also provides lipid nanoparticles comprising at least one nucleic acid molecule, at least one lipidoid, at least one structured lipid, at least one phospholipid, at least one PEGylated lipid, or any combination thereof.

[0184] The lipidoid can be C12-200, also referred to as 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol), hereafter referred to as "C12-200" (see U.S. Pat. Nos. 8,450,298, 8,969,353, 9,556,110, and 10,189,802; see also U.S. Patent Publication No. 2019-0177289).

[0185] Thus, lipid nanoparticles comprising at least one nucleic acid molecule of the present disclosure may comprise about 25 mol% to about 45 mol% C12-200, about 32 mol% to about 52 mol% of at least one structured lipid, about 10 mol% to about 30 mol% of at least one phospholipid, and about 0.1 mol% to about 13 mol% of at least one PEGylated lipid. In some embodiments, lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 30 mol% to about 40 mol% C12-200, about 37 mol% to about 47 mol% of at least one structured lipid, about 15 mol% to about 25 mol% of at least one phospholipid, and about 0.1 mol% to about 8 mol% of at least one PEGylated lipid. In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 32.5 mol% to about 37.5 mol% C12-200, about 39.5 mol% to about 44.5 mol% of at least one structured lipid, about 17.5 mol% to about 22.5 mol% of at least one phospholipid, and about 0.5 mol% to about 5.5 mol% of at least one PEGylated lipid. In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 35 mol% C12-200, about 42 mol% of at least one structured lipid, about 20 mol% of at least one phospholipid, and about 3 mol% of at least one PEGylated lipid. In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 35 mol% C12-200, about 41.84 mol% of at least one structured lipid, about 20 mol% of at least one phospholipid, and about 3.16 mol% of at least one PEGylated lipid. In some embodiments, the lipid nanoparticles may comprise lipids and nucleic acids at a lipid:nucleic acid weight / weight ratio of about 60:1 to about 100:1, or about 70:1 to about 90:1, or about 75:1 to about 85:1. In some embodiments, the lipid nanoparticles may comprise lipids and nucleic acids at a lipid:nucleic acid weight / weight ratio of about 80:1. The phospholipid may be DOPE. The phospholipid may be DPSC. The phospholipid may be DOPC. The structured lipid may be cholesterol. The PEGylated lipid may be DMG-PEG2000.

[0186] In some embodiments, the at least one nucleic acid molecule is a DNA molecule or an RNA molecule. In some embodiments, the nucleic acid molecule is a DNA molecule (e.g., a DoggyBone DNA molecule). Thus, the present disclosure provides a lipid nanoparticle comprising about 35 mol% C12-200, about 42 mol% at least one structured lipid, about 20 mol% at least one phospholipid, and about 3 mol% at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. The present disclosure also provides a lipid nanoparticle comprising about 35 mol% C12-200, about 41.84 mol% at least one structured lipid, about 20 mol% at least one phospholipid, and about 3.16 mol% at least one PEGylated lipid, wherein the at least one nucleic acid comprises at least one DNA molecule. In some embodiments, the at least one DNA molecule is a DoggyBone DNA molecule. In some embodiments, the at least one DNA molecule is a DNA nanoplasmid. In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 35 mol% of at least one titratable cationic lipid, about 42 mol% of at least one structural lipid, about 20 mol% of at least one phospholipid, and about 3 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid is a DNA molecule (e.g., DoggyBone DNA molecule, DNA Nanoplasmid), and the ratio of lipid to nucleic acid in the nanoparticle is about 80:1 (weight / weight). In some embodiments, the lipid nanoparticles comprising at least one nucleic acid molecule may comprise about 35 mol% of at least one titratable cationic lipid, about 41.84 mol% of at least one structural lipid, about 20 mol% of at least one phospholipid, and about 3.16 mol% of at least one PEGylated lipid, wherein the at least one nucleic acid is a DNA molecule (e.g., DoggyBone DNA molecule, DNA Nanoplasmid), and the ratio of lipid to nucleic acid in the nanoparticle is about 80:1 (weight / weight). In some embodiments, the lipid nanoparticles described above can be used to deliver at least one DNA molecule (e.g., DoggyBone DNA molecule, DNA nanoplasmid) to at least one liver cell. The phospholipid can be DOPE. The phospholipid can be DPSC.The phospholipid can be DOPC. The structural lipid can be cholesterol. The PEGylated lipid can be DMG-PEG2000.

[0187] Alternative LNP embodiments of the present disclosure Embodiment 1. A composition comprising: about 23 mol% to about 33 mol% of ssPalmO-Ph-P4C2; about 55 mol % to about 65 mol % cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 7 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 2. At least one lipid nanoparticle comprises: about 25.5 mol% to about 30.5 mol% of ssPalmO-Ph-P4C2; about 57.5 mol % to about 62.5 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; and about 0.1 mol % to about 4.5 mol % of DMG-PEG2000. Embodiment 3. At least one lipid nanoparticle comprises: about 27 mol% to about 29 mol% of ssPalmO-Ph-P4C2; about 59 mol% to about 61 mol% cholesterol; About 9 mol% to about 11 mol% DOPE; and about 1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 4. At least one lipid nanoparticle comprises: Approximately 28 mol% of ssPalmO-Ph-P4C2; About 60 mol% cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 1-3, comprising about 2 mol % DMG-PEG2000. Embodiment 5. The composition of any one of embodiments 1 to 4, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 5:1 to about 15:1 (w / w). Embodiment 6. The composition of any one of embodiments 1-5, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 10:1 (w / w). Embodiment 7. The composition of any one of embodiments 1 to 6, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 8. The composition of any one of embodiments 1 to 7, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 100:1 (w / w). Embodiment 9. At least one lipid nanoparticle comprises: Approximately 28 mol% of ssPalmO-Ph-P4C2; About 60 mol% cholesterol; About 10 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of embodiments 1-8, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 10:1 (w / w). Embodiment 10. At least one lipid nanoparticle comprises: Approximately 28 mol% of ssPalmO-Ph-P4C2; About 60 mol% cholesterol; About 10 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of any one of embodiments 1-9, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 11. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 24.5 mol % to about 34.5 mol % cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 5.5 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 12. At least one lipid nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 27 mol % to about 32 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; and about 0.1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 13. At least one lipid nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 28.5 mol % to about 30.5 mol % cholesterol; About 9 mol% to about 11 mol% DOPE; and about 0.1 mol % to about 1.5 mol % of DMG-PEG2000. Embodiment 14. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 29.5 mol % cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 11-13, comprising about 0.5 mol % DMG-PEG2000. Embodiment 15. The composition of any one of embodiments 11 to 14, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 16. The composition of any one of embodiments 11 to 15, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 17. At least one nanoparticle comprises: Approximately 60 mol% Coatsome SS-OP; about 29.5 mol % cholesterol; About 10 mol % DOPE; about 0.5 mol % DMG-PEG2000; The composition of any one of embodiments 11-16, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 18. A composition comprising: about 27.92 mol% to about 37.92 mol% of ssPalmO-Ph-P4C2; about 27.92 mol % to about 37.92 mol % cholesterol; about 27.92 mol% to about 37.92 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.25 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 19. At least one lipid nanoparticle comprises: about 30.42 mol% to about 35.42 mol% of ssPalmO-Ph-P4C2; about 30.42 mol % to about 35.42 mol % cholesterol; About 30.42 mol% to about 35.42 mol% DOPE; The composition of embodiment 18, comprising about 0.1 mol % to about 3.75 mol % of DMG-PEG2000. Embodiment 20. At least one lipid nanoparticle comprises: about 31.92 mol% to about 32.92 mol% of ssPalmO-Ph-P4C2; about 31.92 mol % to about 32.92 mol % cholesterol; about 31.92 mol% to about 32.92 mol% DOPE; and about 0.25 mol % to about 2.25 mol % of DMG-PEG2000. Embodiment 21. At least one lipid nanoparticle comprises: about 32.92 mol% of ssPalmO-Ph-P4C2; about 32.92 mol % cholesterol; about 32.92 mol % DOPE; 21. The composition of any one of embodiments 18-20, comprising about 1.25 mol % DMG-PEG2000. Embodiment 22. The composition of any one of embodiments 18 to 21, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 50:1 to about 60:1 (w / w). Embodiment 23. The composition of any one of embodiments 18 to 22, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 55:1 (w / w). Embodiment 24. At least one lipid nanoparticle comprises: about 32.92 mol% of ssPalmO-Ph-P4C2; about 32.92 mol % cholesterol; about 32.92 mol % DOPE; about 1.25 mol % DMG-PEG2000; The composition of any one of embodiments 18-23, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 55:1 (w / w). Embodiment 25. A composition comprising: about 5 mol% to about 15 mol% of ssPalmO-Ph-P4C2; about 24.5 mol % to about 34.5 mol % cholesterol; about 55 mol% to about 65 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 5.5 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 26. At least one lipid nanoparticle comprises: about 7.5 mol% to about 12.5 mol% of ssPalmO-Ph-P4C2; about 27 mol % to about 32 mol % cholesterol; about 57.5 mol % to about 62.5 mol % DOPE; The composition of embodiment 25, comprising about 0.1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 27. At least one lipid nanoparticle comprises: about 9 mol% to about 11 mol% of ssPalmO-Ph-P4C2; about 28.5 mol % to about 30.5 mol % cholesterol; about 59 mol% to about 61 mol% DOPE; and about 0.1 mol % to about 1.5 mol % of DMG-PEG2000. Embodiment 28. At least one lipid nanoparticle comprises: about 10 mol% of ssPalmO-Ph-P4C2; about 29.5 mol % cholesterol; About 60 mol% DOPE; The composition of any one of embodiments 25-27, comprising about 0.5 mol % DMG-PEG2000. Embodiment 29. The composition of any one of embodiments 25 to 28, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 30. The composition of any one of embodiments 25 to 29, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1. Embodiment 31. At least one lipid nanoparticle comprises: about 10 mol% of ssPalmO-Ph-P4C2; about 29.5 mol % cholesterol; About 60 mol% DOPE; about 0.5 mol % DMG-PEG2000; The composition of any one of embodiments 25 to 30, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 32. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 23 mol% to about 33 mol% cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.1 mol% to about 7 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 33. At least one lipid nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 25.5 mol % to about 30.5 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; The composition of embodiment 32, comprising about 0.1 mol % to about 4.5 mol % of DMG-PEG2000. Embodiment 34. At least one lipid nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 27 mol% to about 29 mol% cholesterol; About 9 mol% to about 11 mol% DOPE; The composition of embodiment 32 or embodiment 33, comprising about 1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 35. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; About 28 mol % cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 32-34, comprising about 2 mol % DMG-PEG2000. Embodiment 36. The composition of any one of embodiments 32 to 35, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 37. The composition of any one of embodiments 32 to 36, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 38. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; About 28 mol % cholesterol; About 10 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of any one of embodiments 32-27, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 39. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 23.75 mol % to about 33.75 mol % cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.25 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 40. At least one nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 26.25 mol % to about 31.25 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; The composition of embodiment 39, comprising about 0.1 mol % to about 3.75 mol % of DMG-PEG2000. Embodiment 41. At least one nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 27.75 mol % to about 29.75 mol % cholesterol; About 9 mol% to about 11 mol% DOPE; The composition of embodiment 39 or embodiment 40, comprising about 0.25 mol % to about 2.25 mol % of DMG-PEG2000. Embodiment 42. At least one nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 28.75 mol % cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 39-41, comprising about 1.25 mol % DMG-PEG2000. Embodiment 43. The composition of any one of embodiments 39 to 42, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 44. The composition of any one of embodiments 39 to 43, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 100:1 (w / w). Embodiment 45. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 28.75 mol % cholesterol; About 10 mol % DOPE; about 1.25 mol % DMG-PEG2000; The composition of any one of embodiments 39-44, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 46. A composition comprising: about 17.71 mol% to about 27.71 mol% of ssPalmO-Ph-P4C2; about 50.21 mol % to about 60.21 mol % cholesterol; about 23.83 mol% to about 33.83 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.25 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the at least one nucleic acid molecule comprises at least one DNA molecule. Embodiment 47. At least one lipid nanoparticle comprises: about 20.21 mol% to about 25.21 mol% of ssPalmO-Ph-P4C2; about 52.71 mol % to about 57.71 mol % cholesterol; about 26.33 mol% to about 31.33 mol% DOPE; The composition of embodiment 46, comprising about 0.1 mol % to about 3.75 mol % of DMG-PEG2000. Embodiment 48. At least one lipid nanoparticle comprises: about 21.71 mol% to about 23.71 mol% of ssPalmO-Ph-P4C2; about 54.21 mol % to about 56.21 mol % cholesterol; about 27.83 mol% to about 29.83 mol% DOPE; The composition of embodiment 46 or embodiment 47, comprising about 0.25 mol % to about 2.25 mol % of DMG-PEG2000. Embodiment 49. At least one lipid nanoparticle comprises: Approximately 22.71 mol% of ssPalmO-Ph-P4C2; about 55.21 mol % cholesterol; about 28.83 mol % DOPE; The composition of any one of embodiments 46-48, comprising about 1.25 mol % DMG-PEG2000. Embodiment 50. The composition of any one of embodiments 46 to 49, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 51. The composition of any one of embodiments 46 to 50, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 52. At least one lipid nanoparticle comprises: Approximately 22.71 mol% of ssPalmO-Ph-P4C2; about 55.21 mol % cholesterol; about 28.83 mol % DOPE; about 1.25 mol % DMG-PEG2000; The composition of any one of embodiments 46-51, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 53. A composition comprising: about 45 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 33.6 mol % to about 43.6 mol % cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.4 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the at least one nucleic acid molecule comprises at least one DNA molecule. Embodiment 54. At least one lipid nanoparticle comprises: about 47.5 mol% to about 52.5 mol% of ssPalmO-Ph-P4C2; about 36.1 mol % to about 41.1 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; The composition of embodiment 53, comprising about 0.1 mol % to about 3.9 mol % of DMG-PEG2000. Embodiment 55. At least one lipid nanoparticle comprises: about 49 mol% to about 51 mol% of ssPalmO-Ph-P4C2; about 37.6 mol % to about 39.6 mol % cholesterol; About 9 mol% to about 11 mol% DOPE; The composition of embodiment 53 or embodiment 54, comprising about 0.4 mol % to about 2.4 mol % of DMG-PEG2000. Embodiment 56. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, about 38.6 mol % cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 53-55, comprising about 1.4 mol % DMG-PEG2000. Embodiment 57. The composition of any one of embodiments 53 to 56, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 195:1 to about 205:1 (w / w). Embodiment 58. The composition of any one of embodiments 53 to 57, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 200:1 (w / w). Embodiment 59. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, about 38.6 mol % cholesterol; About 10 mol % DOPE; about 1.4 mol % DMG-PEG2000; The composition of any one of embodiments 53-58, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 200:1 (w / w). Embodiment 60. A composition comprising: about 29.69 mol% to about 39.69 mol% of ssPalmO-Ph-P4C2; about 34.74 mol % to about 44.74 mol % cholesterol; about 19.69 mol% to about 29.69 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.25 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the at least one nucleic acid molecule comprises at least one DNA molecule. Embodiment 61. At least one lipid nanoparticle comprises: about 32.19 mol% to about 37.19 mol% of ssPalmO-Ph-P4C2; about 37.24 mol % to about 42.24 mol % cholesterol; about 22.19 mol% to about 27.19 mol% DOPE; The composition of embodiment 60, comprising about 0.1 mol % to about 3.75 mol % of DMG-PEG2000. Embodiment 62. At least one lipid nanoparticle comprises: about 33.69 mol% to about 35.69 mol% of ssPalmO-Ph-P4C2; about 38.74 mol % to about 40.74 mol % cholesterol; about 23.69 mol% to about 25.69 mol% DOPE; The composition of embodiment 60 or embodiment 61, comprising about 0.25 mol % to about 2.25 mol % of DMG-PEG2000. Embodiment 63. At least one lipid nanoparticle comprises: about 34.69 mol% of ssPalmO-Ph-P4C2; about 39.74 mol % cholesterol; about 24.69 mol % DOPE; The composition of any one of embodiments 60-62, comprising about 1.25 mol % DMG-PEG2000. Embodiment 64. The composition of any one of embodiments 60 to 63, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 45:1 to about 55:1 (w / w). Embodiment 65. The composition of any one of embodiments 60 to 64, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 50:1 (w / w). Embodiment 66. At least one lipid nanoparticle comprises: about 34.69 mol% of ssPalmO-Ph-P4C2; about 39.74 mol % cholesterol; about 24.69 mol % DOPE; about 1.25 mol % DMG-PEG2000; The composition of any one of embodiments 60-65, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 50:1 (w / w). Embodiment 67. A composition comprising: about 45 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 15 mol % to about 25 mol % cholesterol; about 24.5 mol% to about 34.5 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 5.5 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the at least one nucleic acid molecule comprises at least one DNA molecule. Embodiment 68. At least one lipid nanoparticle comprises: about 47.5 mol% to about 52.5 mol% of ssPalmO-Ph-P4C2; about 17.5 mol % to about 22.5 mol % cholesterol; About 27 mol% to about 32 mol% DOPE; The composition of embodiment 67, comprising about 0.1 mol% to about 3 mol% of DMG-PEG2000. Embodiment 69. At least one lipid nanoparticle comprises: about 49 mol% to about 51 mol% of ssPalmO-Ph-P4C2; about 19 mol% to about 21 mol% cholesterol; About 28.5 mol% to about 30.5 mol% DOPE; The composition of embodiment 67 or embodiment 68, comprising about 0.25 mol % to about 1.5 mol % of DMG-PEG2000. Embodiment 70. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, About 20 mol % cholesterol; about 29.5 mol % DOPE; The composition of any one of embodiments 67-69, comprising about 0.5 mol % DMG-PEG2000. Embodiment 71. The composition of any one of embodiments 67 to 70, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 72. The composition of any one of embodiments 67 to 71, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 73. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, About 20 mol % cholesterol; about 29.5 mol % DOPE; about 0.5 mol % DMG-PEG2000; The composition of any one of embodiments 67-71, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 74. A composition comprising: about 49 mol% to about 59 mol% of ssPalmO-Ph-P4C2; About 30 mol % to about 40 mol % cholesterol; About 5 mol% to about 15 mol% of DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 75. At least one lipid nanoparticle comprises: about 51.5 mol% to about 56.5 mol% of ssPalmO-Ph-P4C2; about 32.5 mol % to about 37.5 mol % cholesterol; About 7.5 mol% to about 12.5 mol% DOPE; The composition of embodiment 74, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 76. At least one lipid nanoparticle comprises: about 53 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 59 mol% to about 61 mol% cholesterol; About 9 mol% to about 11 mol% DOPE; The composition of embodiment 74 or embodiment 75, comprising about 0.5 mol % to about 2 mol % of DMG-PEG2000. Embodiment 77. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; The composition of any one of embodiments 74-76, comprising about 1 mol % DMG-PEG2000. Embodiment 78. The composition of any one of embodiments 74 to 77, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 79. The composition of any one of embodiments 74 to 78, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 100:1 (w / w). Embodiment 80. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; about 1 mol % DMG-PEG2000; The composition of any one of embodiments 74-79, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 81. A composition comprising: about 22.84 mol% to about 32.84 mol% of ssPalmO-Ph-P4C2; about 51.25 mol % to about 61.25 mol % cholesterol; About 8.46 mol% to about 18.46 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 7.45 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 82. At least one lipid nanoparticle comprises: about 25.34 mol% to about 30.34 mol% of ssPalmO-Ph-P4C2; about 53.75 mol % to about 58.75 mol % cholesterol; about 10.96 mol% to about 15.96 mol% DOPE; The composition of embodiment 81, comprising about 0.1 mol% to about 4.95 mol% of DMG-PEG2000. Embodiment 83. At least one lipid nanoparticle comprises: about 26.84 mol% to about 28.84 mol% of ssPalmO-Ph-P4C2; about 55.25 mol % to about 57.25 mol % cholesterol; about 12.46 mol% to about 13.46 mol% DOPE; The composition of embodiment 81 or embodiment 82, comprising about 1.45 mol % to about 3.45 mol % of DMG-PEG2000. Embodiment 84. At least one lipid nanoparticle comprises: about 27.84 mol% of ssPalmO-Ph-P4C2; about 56.25 mol % cholesterol; about 13.46 mol % DOPE; The composition of any one of embodiments 81-83, comprising about 2.45 mol % DMG-PEG2000. Embodiment 85. The composition of any one of embodiments 81 to 84, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 83:1 to about 93:1 (w / w). Embodiment 86. The composition of any one of embodiments 81 to 85, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 88:1 (w / w). Embodiment 87. At least one lipid nanoparticle comprises: about 27.84 mol% of ssPalmO-Ph-P4C2; about 56.25 mol % cholesterol; about 13.46 mol % DOPE; about 2.45 mol % DMG-PEG2000; The composition of any one of embodiments 81-86, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 88:1 (w / w). Embodiment 88. A composition comprising: about 22.9 mol% to about 32.9 mol% of ssPalmO-Ph-P4C2; about 46.51 mol% to about mol% cholesterol; about 13.59 mol% to about 23.59 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 7 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 89. At least one lipid nanoparticle comprises: about 25.4 mol% to about 30.4 mol% of ssPalmO-Ph-P4C2; about 49.01 mol % to about mol % cholesterol; about 16.09 mol% to about 21.09 mol% DOPE; The composition of embodiment 88, comprising about 0.1 mol % to about 4.5 mol % of DMG-PEG2000. Embodiment 90. At least one lipid nanoparticle comprises: about 26.9 mol% to about 28.9 mol% of ssPalmO-Ph-P4C2; about 50.51 mol % to about 52.51 mol % cholesterol; about 17.59 mol% to about 19.59 mol% DOPE; The composition of embodiment 88 or embodiment 89, comprising about 1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 91. At least one lipid nanoparticle comprises: about 27.9 mol% of ssPalmO-Ph-P4C2; about 51.51 mol % cholesterol; about 18.59 mol % DOPE; The composition of any one of embodiments 88-90, comprising about 2 mol % DMG-PEG2000. Embodiment 92. The composition of any one of embodiments 88 to 91, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 93. The composition of any one of embodiments 88 to 92, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 100:1 (w / w). Embodiment 94. At least one lipid nanoparticle comprises: about 27.9 mol% of ssPalmO-Ph-P4C2; about 51.51 mol % cholesterol; about 18.59 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of any one of embodiments 88-93, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 95. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 21.4 mol % to about 31.4 mol % cholesterol; About 6.6 mol% to about 16.6 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 7 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 96. At least one lipid nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 23.9 mol % to about 28.9 mol % cholesterol; About 9.1 mol% to about 14.1 mol% DOPE; The composition of embodiment 95, comprising about 0.1 mol % to about 4.5 mol % of DMG-PEG2000. Embodiment 97. At least one lipid nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 25.4 mol % to about 27.4 mol % cholesterol; About 10.6 mol% to about 12.6 mol% DOPE; The composition of embodiment 95 or embodiment 96, comprising about 1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 98. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 26.4 mol % cholesterol; about 11.6 mol % DOPE; The composition of any one of embodiments 95-97, comprising about 2 mol% DMG-PEG2000. Embodiment 99. The composition of any one of embodiments 95 to 98, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 65:1 to about 75:1 (w / w). Embodiment 100. The composition of any one of embodiments 95 to 99, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 70:1 (w / w). Embodiment 101. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 26.4 mol % cholesterol; about 11.6 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of any one of embodiments 95-100, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 70:1 (w / w). Embodiment 102. A composition comprising: about 25.37 mol% to about 35.37 mol% of ssPalmO-Ph-P4C2; about 32.27 mol % to about 42.27 mol % cholesterol; about 25.36 mol% to about 35.36 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 7 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule. Embodiment 103. At least one lipid nanoparticle comprises: about 27.87 mol% to about 32.87 mol% of ssPalmO-Ph-P4C2; about 34.77 mol % to about 39.77 mol % cholesterol; About 27.86 mol% to about 32.86 mol% DOPE; The composition of embodiment 102, comprising about 0.1 mol % to about 4.5 mol % of DMG-PEG2000. Embodiment 104. At least one lipid nanoparticle comprises: about 29.37 mol% to about 31.37 mol% of ssPalmO-Ph-P4C2; about 36.27 mol % to about 38.27 mol % cholesterol; about 29.36 mol% to about 31.36 mol% DOPE; The composition of embodiment 102 or embodiment 103, comprising about 1 mol % to about 3 mol % of DMG-PEG2000. Embodiment 105. At least one lipid nanoparticle comprises: about 30.37 mol% of ssPalmO-Ph-P4C2; about 37.27 mol % cholesterol; about 30.36 mol % DOPE; The composition of any one of embodiments 102-104, comprising about 2 mol % DMG-PEG2000. Embodiment 106. The composition of any one of embodiments 102 to 105, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 95:1 to about 105:1 (w / w). Embodiment 107. The composition of any one of embodiments 102 to 106, wherein the ratio of lipid to nucleic acid in at least one nanoparticle is about 100:1 (w / w). Embodiment 108. At least one lipid nanoparticle comprises: about 30.37 mol% of ssPalmO-Ph-P4C2; about 37.27 mol % cholesterol; about 30.36 mol % DOPE; about 2 mol % DMG-PEG2000; The composition of any one of embodiments 102-107, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 109. A composition comprising: about 45 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 15 mol % to about 25 mol % cholesterol; about 23.7 mol% to about 33.7 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.3 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and wherein the at least one nucleic acid molecule comprises at least one DNA molecule. Embodiment 110. At least one lipid nanoparticle comprises: about 47.5 mol% to about 52.5 mol% of ssPalmO-Ph-P4C2; about 17.5 mol % to about 22.5 mol % cholesterol; About 26.2 mol% to about 31.2 mol% DOPE; The composition of embodiment 109, comprising about 0.1 mol% to about 4 mol% of DMG-PEG2000. Embodiment 111. At least one lipid nanoparticle comprises: about 49 mol% to about 51 mol% of ssPalmO-Ph-P4C2; about 19 mol% to about 21 mol% cholesterol; about 27.7 mol% to about 29.7 mol% DOPE; The composition of embodiment 109 or embodiment 110, comprising about 0.25 mol % to about 2.3 mol % of DMG-PEG2000. Embodiment 112. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, About 20 mol % cholesterol; about 28.7 mol% DOPE; The composition of any one of embodiments 109-111, comprising about 1.3 mol % DMG-PEG2000. Embodiment 113. The composition of any one of embodiments 109 to 112, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 45:1 to about 55:1 (w / w). Embodiment 114. The composition of any one of embodiments 109 to 113, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 50:1 (w / w). Embodiment 115. At least one lipid nanoparticle comprises: About 50 mol% of ssPalmO-Ph-P4C2, About 20 mol % cholesterol; about 28.7 mol% DOPE; about 1.3 mol % DMG-PEG2000; The composition of any one of embodiments 109-114, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 50:1 (w / w). Embodiment 116. A composition comprising: about 49 mol% to about 59 mol% of ssPalmO-Ph-P4C2; About 30 mol % to about 40 mol % cholesterol; About 5 mol% to about 15 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 117. At least one lipid nanoparticle comprises: about 51.5 mol% to about 56.5 mol% of ssPalmO-Ph-P4C2; about 32.5 mol % to about 37.5 mol % cholesterol; About 7.5 mol% to about 12.5 mol% of a phospholipid; The composition of embodiment 116, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 118. At least one lipid nanoparticle comprises: about 53 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 34 mol% to about 36 mol% cholesterol; About 9 mol% to about 11 mol% of a phospholipid; The composition of embodiment 116 or embodiment 117, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000.

[0188] Embodiment 119. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % of phospholipids; The composition of any one of embodiments 116-118, comprising about 1 mol % DMG-PEG2000. Embodiment 120. A composition comprising: about 54 mol% to about 64 mol% of ssPalmO-Ph-P4C2; About 30 mol % to about 40 mol % cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 121. At least one lipid nanoparticle comprises: about 56.5 mol% to about 61.5 mol% of ssPalmO-Ph-P4C2; about 32.5 mol % to about 37.5 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 120, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 122. At least one lipid nanoparticle comprises: about 58 mol% to about 60 mol% of ssPalmO-Ph-P4C2; about 34 mol% to about 36 mol% cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 120 or embodiment 121, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000. Embodiment 123. At least one lipid nanoparticle comprises: Approximately 59 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 120-122, comprising about 1 mol % DMG-PEG2000. Embodiment 124. A composition comprising: about 49 mol% to about 59 mol% of ssPalmO-Ph-P4C2; about 35 mol % to about 45 mol % cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 125. At least one lipid nanoparticle comprises: about 51.5 mol% to about 56.5 mol% of ssPalmO-Ph-P4C2; about 37.5 mol % to about 42.5 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 124, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 126. At least one lipid nanoparticle comprises: about 53 mol% to about 55 mol% of ssPalmO-Ph-P4C2; about 39 mol% to about 41 mol% cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 124 or embodiment 125, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000. Embodiment 127. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 40 mol% cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 124-126, comprising about 1 mol % DMG-PEG2000. Embodiment 128. A composition comprising: about 51.5 mol% to about 61.5 mol% of ssPalmO-Ph-P4C2; about 32.5 mol % to about 42.5 mol % cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 129. At least one lipid nanoparticle comprises: about 54 mol% to about 59 mol% of ssPalmO-Ph-P4C2; About 35 mol % to about 40 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 128, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 130. At least one lipid nanoparticle comprises: about 55.5 mol% to about 57.5 mol% of ssPalmO-Ph-P4C2; about 36.5 mol % to about 38.5 mol % cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 128 or embodiment 129, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000. Embodiment 131. At least one lipid nanoparticle comprises: about 56.5 mol% of ssPalmO-Ph-P4C2; about 37.5 mol % cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 128-130, comprising about 1 mol % DMG-PEG2000. Embodiment 132. A composition comprising: about 44.4 mol% to about 54.4 mol% of ssPalmO-Ph-P4C2; about 39 mol% to about 49 mol% cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6.6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 133. At least one lipid nanoparticle comprises: about 46.9 mol% to about 51.9 mol% of ssPalmO-Ph-P4C2; about 41.5 mol % to about 46.5 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 132, comprising about 0.1 mol % to about 4.1 mol % of DMG-PEG2000. Embodiment 134. At least one lipid nanoparticle comprises: about 48.4 mol% to about 50.4 mol% of ssPalmO-Ph-P4C2; about 43 mol % to about 45 mol % cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 132 or embodiment 133, comprising about 0.6 mol % to about 2.6 mol % of DMG-PEG2000. Embodiment 135. At least one lipid nanoparticle comprises: Approximately 49.4 mol% of ssPalmO-Ph-P4C2; About 44 mol% cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 132-134, comprising about 1.6 mol % DMG-PEG2000. Embodiment 136. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 27.8 mol % to about 37.8 mol % cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 7.2 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 137. At least one lipid nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 30.3 mol % to about 35.3 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 136, comprising about 0.1 mol % to about 4.7 mol % of DMG-PEG2000. Embodiment 138. At least one lipid nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 31.8 mol % to about 33.8 mol % cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 136 or embodiment 137, comprising about 1.2 mol % to about 2.2 mol % of DMG-PEG2000. Embodiment 139. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 32.8 mol % cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 136-138, comprising about 2.2 mol % DMG-PEG2000. Embodiment 140. A composition comprising: about 55 mol% to about 65 mol% of ssPalmO-Ph-P4C2; about 29 mol% to about 39 mol% cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 141. At least one lipid nanoparticle comprises: about 57.5 mol% to about 62.5 mol% of ssPalmO-Ph-P4C2; about 31.5 mol % to about 36.5 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 140, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 142. At least one lipid nanoparticle comprises: about 59 mol% to about 61 mol% of ssPalmO-Ph-P4C2; about 33 mol % to about 35 mol % cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 140 or embodiment 141, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000. Embodiment 143. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; About 34 mol % cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 140-142, comprising about 1 mol % DMG-PEG2000. Embodiment 144. A composition comprising: about 36.8 mol% to about 46.8 mol% of ssPalmO-Ph-P4C2; about 47.2 mol % to about 57.2 mol % cholesterol; About 0.1 mol% to about 10 mol% of a phospholipid; At least one lipid nanoparticle comprising about 0.01 mol% to about 6 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 145. At least one lipid nanoparticle comprises: about 39.3 mol% to about 44.3 mol% of ssPalmO-Ph-P4C2; about 49.7 mol % to about 54.7 mol % cholesterol; About 2.5 mol% to about 7.5 mol% of a phospholipid; The composition of embodiment 144, comprising about 0.1 mol % to about 3.5 mol % of DMG-PEG2000. Embodiment 146. At least one lipid nanoparticle comprises: about 40.8 mol% to about 42.8 mol% of ssPalmO-Ph-P4C2; about 51.2 mol % to about 53.2 mol % cholesterol; About 4 mol% to about 6 mol% of a phospholipid; The composition of embodiment 144 or embodiment 145, comprising about 0.25 mol % to about 2 mol % of DMG-PEG2000. Embodiment 147. At least one lipid nanoparticle comprises: Approximately 41.8 mol% of ssPalmO-Ph-P4C2; about 52.2 mol % cholesterol; About 5 mol % phospholipids; The composition of any one of embodiments 144-146, comprising about 1 mol % DMG-PEG2000. Embodiment 148. The composition of any one of embodiments 116 to 147, wherein at least one nucleic acid molecule is an RNA molecule. Embodiment 149. The composition of embodiment 148, wherein the RNA molecule is an mRNA molecule. Embodiment 150. The composition of embodiment 149, wherein the mRNA molecule further comprises a 5'-CAP. Embodiment 151. The composition of any one of embodiments 116 to 147, wherein at least one nucleic acid molecule is a DNA molecule. Embodiment 152. The composition of embodiment 151, wherein the DNA molecule is a DoggyBone DNA molecule. Embodiment 153. The composition of embodiment 151, wherein the DNA molecule is a DNA nanoplasmid. Embodiment 154. The composition of any one of embodiments 116 to 153, wherein the phospholipid is DOPE. Embodiment 155. The composition of any one of embodiments 116 to 153, wherein the phospholipid is DOPC. Embodiment 156. The composition of any one of embodiments 116 to 153, wherein the phospholipid is DSPC. Embodiment 157. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 55:1 to about 110:1 (w / w). Embodiment 158. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 20:1 (w / w). Embodiment 159. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 40:1 (w / w). Embodiment 160. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 60:1 (w / w). Embodiment 161. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 75:1 (w / w). Embodiment 162. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 80:1 (w / w). Embodiment 163. The composition of any one of embodiments 116 to 156, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w). Embodiment 164. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 165. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 166. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 167. At least one lipid nanoparticle comprises: Approximately 59 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 5 mol% DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 168. At least one lipid nanoparticle comprises: Approximately 59 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 5 mol% DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 169. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 40 mol% cholesterol; About 5 mol% DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 170. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 40 mol% cholesterol; About 5 mol% DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 171. At least one lipid nanoparticle comprises: about 56.5 mol% of ssPalmO-Ph-P4C2; about 37.5 mol % cholesterol; About 5 mol% DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 172. At least one lipid nanoparticle comprises: about 56.5 mol% of ssPalmO-Ph-P4C2; about 37.5 mol % cholesterol; About 5 mol% DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 173. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w / w). Embodiment 174. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w / w). Embodiment 175. At least one lipid nanoparticle comprises: Approximately 59 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 5 mol% DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 75:1 (w / w). Embodiment 176. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one DNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 100:1 (w / w). Embodiment 177. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPE; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 178. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 179. At least one lipid nanoparticle comprises: Approximately 49.4 mol% of ssPalmO-Ph-P4C2; About 44 mol% cholesterol; About 5 mol% DOPC; about 1.6 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 180. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; about 32.8 mol % cholesterol; About 5 mol% DSPC; about 2.2 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 181. At least one lipid nanoparticle comprises: Approximately 60 mol% of ssPalmO-Ph-P4C2; About 34 mol % cholesterol; About 5 mol% DOPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 40:1 (w / w). Embodiment 182. At least one lipid nanoparticle comprises: Approximately 41.8 mol% of ssPalmO-Ph-P4C2; about 52.2 mol % cholesterol; About 5 mol% DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 183. At least one lipid nanoparticle comprises: Approximately 54 mol% of ssPalmO-Ph-P4C2; About 35 mol % cholesterol; About 10 mol % DSPC; about 1 mol % DMG-PEG2000; The composition of any one of the preceding embodiments, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule, and the at least one nucleic acid molecule comprises at least one RNA molecule, and the ratio of lipid to nucleic acid in the at least one nanoparticle is about 60:1 (w / w). Embodiment 184. The composition of any one of the preceding embodiments, wherein the mRNA comprises a cytidine residue that is 5-methylcytidine (5-MeC). Embodiment 185. A composition comprising: about 30 mol% to about 40 mol% C12-200; about 36.84 mol % to about 46.84 mol % cholesterol; about 15 mol% to about 25 mol% DOPE; At least one lipid nanoparticle comprising about 0.01 mol% to about 8.16 mol% DMG-PEG2000; A composition, wherein at least one lipid nanoparticle comprises at least one nucleic acid molecule. Embodiment 186. At least one lipid nanoparticle comprises: about 32.5 mol % to about 37.5 mol % C12-200; about 39.34 mol % to about 44.34 mol % cholesterol; about 17.5 mol % to about 22.5 mol % DOPE; The composition of embodiment 185, comprising about 0.1 mol% to about 5.66 mol% DMG-PEG2000. Embodiment 187. At least one lipid nanoparticle comprises: about 34 mol% to about 36 mol% C12-200; about 40.84 mol % to about 42.84 mol % cholesterol; about 19 mol% to about 21 mol% DOPE; The composition of embodiment 185 or embodiment 186, comprising about 2.16 mol % to about 4.16 mol % of DMG-PEG2000. Embodiment 188. At least one lipid nanoparticle comprises: About 35 mol% C12-200; about 41.84 mol % cholesterol; About 20 mol % DOPE; The composition of any one of embodiments 185-187, comprising about 3.16 mol % DMG-PEG2000. Embodiment 189. The composition of any one of embodiments 185 to 188, wherein the at least one nucleic acid molecule is at least one DNA molecule. Embodiment 190. The composition of embodiment 189, wherein the DNA molecule is a DoggyBone DNA molecule. Embodiment 191. The composition of embodiment 190, wherein the DNA molecule is a DNA nanoplasmid. Embodiment 192. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is from about 70:1 to about 90:1 (w / w). Embodiment 193. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 20:1 (w / w). Embodiment 194. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 40:1 (w / w). Embodiment 195. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 60:1 (w / w). Embodiment 196. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 75:1 (w / w). Embodiment 197. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 80:1 (w / w). Embodiment 198. The composition of any one of embodiments 185 to 191, wherein the ratio of lipid to nucleic acid in at least one lipid nanoparticle is about 100:1 (w / w).

[0189] Pharmaceutical Compositions of the Present Disclosure In some aspects, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure.

[0190] In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one lipid nanoparticle of the present disclosure.In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one first nanoparticle of the present disclosure and at least one second nanoparticle of the present disclosure, wherein at least one first nanoparticle comprises at least one nucleic acid molecule encoding at least one transposase, and at least one second nanoparticle comprises at least one nucleic acid molecule encoding at least one transposon.

[0191] In some embodiments, the present disclosure provides a composition comprising at least one cell that is contacted with at least one lipid nanoparticle of the present disclosure.In some embodiments, the present disclosure provides a composition comprising at least one cell that is genetically modified using at least one lipid nanoparticle of the present disclosure.In some embodiments, the present disclosure provides a composition comprising at least one cell that is genetically modified using any method of the present disclosure.

[0192] Methods of the Disclosure The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for delivering at least one nucleic acid to at least one cell, comprising contacting the at least one cell with at least one nanoparticle of the present disclosure.

[0193] In all of the methods, compositions, and kits of the present disclosure, at least one cell can be a liver cell, which can include, but is not limited to, a hepatocyte, a hepatic stellate cell, a Kupffer cell, or a hepatic sinusoidal endothelial cell.

[0194] In some aspects of any of the methods of the present disclosure, the cell may be in vivo, ex vivo, or in vitro. In some aspects, any of the methods of the present disclosure may be applied in vivo, ex vivo, or in vitro.

[0195] The present disclosure provides a method for genetically modifying at least one cell, comprising contacting at least one cell with at least one composition of the present disclosure.The present disclosure provides a method for genetically modifying at least one cell, comprising contacting at least one cell with at least one lipid nanoparticle of the present disclosure.

[0196] In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to a cell such that the cell expresses at least one protein that the cell does not otherwise normally express, or such that the cell expresses at least one protein at a level higher than the level at which the cell otherwise normally expresses the at least one protein, or such that the cell expresses at least one protein at a level lower than the level at which the cell otherwise normally expresses the at least one protein. In some embodiments, genetically modifying a cell can include delivering at least one exogenous nucleic acid to a cell such that the at least one exogenous nucleic acid is integrated into the genome of the at least one cell.

[0197] In some embodiments, contacting at least one cell with at least one lipid nanoparticle of the present disclosure may result in at least one cell expressing at least one exogenous protein at a level that is increased by at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, at least 15-fold, or at least about 20-fold, or at least about 25-fold, or at least about 30-fold, or at least about 50-fold, compared to the expression level of the exogenous protein induced by contacting at least one cell with at least one control lipid nanoparticle.

[0198] In some aspects, the methods of the present disclosure can produce a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the plurality of cells express at least one protein encoded by at least one nucleic acid delivered to the plurality of cells via a nanoparticle of the present disclosure.

[0199] In some aspects, the methods of the disclosure may produce a plurality of cells, wherein at least about 1%, or at least about 2%, or at least about 3%, or at least about 4%, or at least about 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 35%, or at least 40%, or at least 45%, or at least 50%, or at least 55%, or at least 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells.

[0200] In some aspects, the nucleic acid molecules formulated in the lipid nanoparticles of the present disclosure may comprise at least one genome editing composition.

[0201] The gene editing composition may comprise at least one nucleic acid molecule comprising at least one nucleic acid sequence encoding a DNA binding domain and a nucleic acid sequence encoding a nuclease protein or a nuclease domain thereof. The nucleic acid sequence encoding the nuclease protein or the sequence encoding the nuclease domain thereof may comprise a DNA sequence, an RNA sequence, or a combination thereof.

[0202] In some aspects, the genome editing compositions formulated in lipid nanoparticles of the present disclosure can include a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising a nuclease-inactivated Cas (dCas) protein or a nuclease domain thereof, and an endonuclease protein or a nuclease domain thereof.

[0203] In some embodiments, the genome editing composition formulated in the lipid nanoparticles of the present disclosure may include a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof, and (ii) a Clo051 protein or a nuclease domain thereof. In some embodiments, the fusion protein may further comprise at least one nuclear localization signal (NLS). In some embodiments, the fusion protein may further comprise at least two NLSs. In some embodiments, the nucleic acid molecule may comprise DNA, RNA, or any combination thereof. In some embodiments, the nucleic acid molecule may comprise RNA. Exemplary dCas9-Clo051 fusion proteins (referred to in the art as "Cas-CLOVER" proteins) and polynucleotide sequences encoding the dCas9-Clo051 fusion proteins are described in detail in U.S. Patent Publication No. 2022 / 0042038, the contents of which are incorporated herein by reference in their entirety. Gene editing compositions including Cas-CLOVER and methods of using these compositions for gene editing are described in detail in U.S. Patent Publication Nos. 2017 / 0107541, 2017 / 0114149, 2018 / 0187185, and U.S. Patent No. 10,415,024, the contents of each of which are incorporated by reference in their entirety herein.

[0204] In some embodiments, the Cas-CLOVER protein may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:31.

[0205] In some embodiments, the Clo051 protein or nuclease domain may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:32.

[0206] In some embodiments, an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof can comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:33.

[0207] In some embodiments, the genome editing composition formulated in the lipid nanoparticles of the present disclosure may further comprise at least one guide molecule. In some embodiments, the guide molecule may be a guide RNA (gRNA molecule).

[0208] Thus, the present disclosure provides any of the lipid nanoparticle compositions described herein, wherein the lipid nanoparticle comprises at least one genome editing composition, the at least one genome editing composition comprising: a) a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof, and b) at least one gRNA molecule. In some embodiments, the fusion protein may further comprise at least one NLS. In some embodiments, the at least one genome editing composition may comprise at least two gRNA molecules.

[0209] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject at least one therapeutically effective amount of at least one composition of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.

[0210] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of cells, the cells being in contact with at least one nanoparticle of the present disclosure comprising at least one nucleic acid encoding a therapeutic protein.The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering at least one therapeutically effective amount of cells, the cells being genetically modified using the compositions and / or methods of the present disclosure.

[0211] In some embodiments, at least one disease can be metabolic liver disease (MLD).MLD can be, but is not limited to, N-acetylglutamate synthetase (NAGS) deficiency, carbamoyl phosphate synthetase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinate synthetase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinate lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), methylmalonic acidemia (MMA), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

[0212] In some embodiments, at least one disease may be a hemophilia disease. In some embodiments, the hemophilia disease is hemophilia A. In some embodiments, the hemophilia disease is hemophilia B. In some embodiments, the hemophilia disease is hemophilia C.

[0213] In some embodiments, the at least one disease can be a disease and / or disorder characterized by elevated LDL cholesterol. Accordingly, the present disclosure provides methods of reducing LDL cholesterol in a subject in need thereof.

[0214] In some embodiments, the nucleic acid molecule formulated in lipid nanoparticles of the present disclosure can comprise at least one transgene sequence.In some embodiments, the transgene sequence can comprise the nucleotide sequence that codes for at least one therapeutic protein.In some embodiments, the transgene sequence can comprise the nucleotide sequence that codes for at least one transposase.

[0215] In some embodiments, the transgene sequence may comprise a nucleotide sequence encoding at least one transposon. In some embodiments, the transposon may comprise a nucleotide sequence encoding at least one therapeutic protein. In some embodiments, the transposon may comprise a nucleotide sequence encoding at least one therapeutic protein and at least one protomer sequence, where the at least one therapeutic protein is operably linked to at least one promoter sequence. In some embodiments, the transposon may comprise at least one inverted terminal repeat (ITR). In some embodiments, the transposon may comprise a first ITR and at least a second ITR. In some embodiments, the transposon may comprise at least one insulator sequence. In some embodiments, the transposon may comprise a first insulator sequence and at least a second insulator sequence. In some embodiments, the transposon may comprise at least one sequence encoding at least one therapeutic protein. In some embodiments, the transposon may comprise at least one 5'UTR sequence. In some embodiments, the transposon may comprise at least one 3'UTR sequence. In some embodiments, the transposon may comprise a first 3'UTR sequence and at least a second 3'UTR sequence. In some embodiments, the transposon may comprise at least one polyA sequence.

[0216] In some embodiments, the transposon may comprise at least one sequence encoding a therapeutic protein and a 3'UTR sequence. In some embodiments, the transposon may comprise, from the 5' to 3' direction, at least one sequence encoding a therapeutic protein and a 3'UTR sequence. In some embodiments, the transposon may comprise at least one sequence encoding a therapeutic protein followed by a 3'UTR sequence.

[0217] In some embodiments, the transposon may comprise a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a 3'UTR sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some embodiments, the transposon may comprise, from 5' to 3', a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a 3'UTR sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some embodiments, the transposon may comprise a first ITR, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one promoter sequence, followed by at least one sequence encoding at least one therapeutic protein, followed by a 3'UTR sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second ITR.

[0218] In some embodiments of the above transposons, at least one sequence encoding at least one therapeutic protein may be a sequence encoding a FVIII-BDD polypeptide, wherein the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the sequence encoding the FVIII-BDD polypeptide may comprise the nucleic acid sequence of SEQ ID NO: 22.

[0219] In some embodiments of the above transposons, the 3'UTR sequence may comprise the nucleic acid sequence of SEQ ID NO:27.

[0220] Thus, in a non-limiting example, the transposon can comprise at least one sequence encoding a therapeutic protein and a 3'UTR sequence, where the at least one sequence encoding a therapeutic protein is a sequence encoding a FVIII-BDD polypeptide, where the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO:21, and the 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:27.

[0221] In some embodiments, the transposon may comprise at least one sequence encoding a therapeutic protein, a first 3'UTR sequence, and a second 3'UTR sequence. In some embodiments, the transposon may comprise, in a 5' to 3' sequence, at least one sequence encoding a therapeutic protein, a first 3'UTR sequence, and a second 3'UTR sequence. In some embodiments, the transposon may comprise at least one sequence encoding a therapeutic protein, followed by a first 3'UTR sequence, followed by a second 3'UTR sequence.

[0222] In some embodiments, the transposon may comprise a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a first 3'UTR sequence, a second 3'UTR sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some embodiments, the transposon may comprise, from 5' to 3', a first ITR, a first insulator sequence, at least one promoter sequence, at least one sequence encoding at least one therapeutic protein, a first 3'UTR sequence, a second 3'UTR sequence, a polyA sequence, a second insulator sequence, and a second ITR. In some embodiments, the transposon may comprise a first ITR, followed by a first insulator sequence, followed by at least one promoter sequence, followed by at least one promoter sequence, followed by at least one sequence encoding at least one therapeutic protein, followed by a first 3'UTR sequence, followed by a second 3'UTR sequence, followed by a polyA sequence, followed by a second insulator sequence, followed by a second ITR.

[0223] In some embodiments of the above transposons, the at least one sequence encoding at least one therapeutic protein may be a sequence encoding a FVIII-BDD polypeptide, the FVIII-BDD polypeptide comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the ta sequence encoding the FVIII-BDD polypeptide may comprise the nucleic acid sequence of SEQ ID NO: 22.

[0224] In some embodiments of the above transposons, the first 3'UTR sequence may be an AES 3'UTR sequence, wherein the AES 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:25.

[0225] In some embodiments of the above transposons, the second 3'UTR sequence may be an mtRNR1 3'UTR sequence, wherein the mtRNR1 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:26.

[0226] Thus, in a non-limiting example, the transposon can comprise at least one sequence encoding a therapeutic protein, a first 3'UTR sequence, and a second 3'UTR sequence, where the at least one sequence encoding a therapeutic protein is a sequence encoding a FVIII-BDD polypeptide, where the FVIII-BDD polypeptide comprises the amino acid sequence of SEQ ID NO:21, the first 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:25, and the second 3'UTR sequence comprises the nucleic acid sequence of SEQ ID NO:26.

[0227] In some embodiments, the transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:28.

[0228] In some embodiments, the transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:34.

[0229] In some embodiments, the transposon may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:35.

[0230] In some embodiments, a therapeutic protein may comprise, consist essentially of, or consist of a methylmalonyl-CoA mutase (MUT1) polypeptide.

[0231] In some embodiments, the therapeutic protein may comprise, consist essentially of, or consist of an ornithine transcarbamylase (OTC) polypeptide.

[0232] In some embodiments, the therapeutic protein may comprise, consist essentially of, or consist of a factor VIII (FVIII) polypeptide. In some embodiments, the FVIII polypeptide may be a FVIII polypeptide lacking the B domain (hereinafter referred to as a FVIII-BDD polypeptide). As will be appreciated by those skilled in the art, factor VIII-BDD polypeptides retain biological activity in vitro and in vivo (see Kessler et al. Haemophilia, 2005, 11(2):84-91).

[0233] In some embodiments, a FVIII-BDD polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 21. In some embodiments, a nucleic acid sequence encoding a FVIII-BDD polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NO: 22 or 36.

[0234] In some embodiments, the therapeutic protein may comprise, consist essentially of, or consist of a factor IX (FIX) polypeptide. In some embodiments, the FIX polypeptide may comprise an R338L mutation. As will be understood by those skilled in the art, the R338L mutation may be referred to as a Padua mutation (see VandenDriessche and Chuah, Molecular Therapy, 2018, Vol. 26, Issue 1, P14-16, the contents of which are incorporated herein by reference in their entirety).

[0235] The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) a therapeutically effective amount of at least one composition comprising a nucleic acid molecule comprising a transposon, where the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) a therapeutically effective amount of at least one composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0236] In some embodiments of the above methods, the composition comprising a nucleic acid molecule comprising a transposon can be a composition comprising at least one LNP of the present disclosure, the LNP comprising at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein. The present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject a) at least one therapeutically effective amount of an LNP of the present disclosure, the LNP comprising at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein, and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0237] In some embodiments of the above method, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be a composition comprising at least one LNP of the present disclosure, where the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase. Thus, the present disclosure provides a method for treating at least one disease in a subject, the method comprising administering to the subject a) at least one therapeutically effective amount of a composition comprising a nucleic acid comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein, and b) at least one therapeutically effective amount of the LNP of the present disclosure, the LNP comprising at least one nucleic acid comprising a nucleotide sequence encoding at least one transposase.

[0238] Additionally, the present disclosure also provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0239] In some embodiments of the above method, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposon can be a composition comprising an adeno-associated virus (AAV) virus vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein.Thus, the present disclosure provides a method for treating at least one disease in a subject, the method comprising administering to the subject a) at least one therapeutically effective amount of an AAV virus vector particle comprising at least one nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, and b) at least one therapeutically effective amount of a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0240] Additionally, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, where the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, where the LNP comprises at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0241] In some embodiments of the above method, the composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase can be a composition comprising an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.Thus, the present disclosure provides a method for treating at least one disease in a subject, the method comprising administering to the subject a) at least one therapeutically effective amount of a composition comprising a nucleic acid comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein, and b) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0242] Additionally, the present disclosure provides a method of treating at least one disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of an LNP of the present disclosure, wherein the LNP comprises at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase.

[0243] In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, where the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, where the therapeutic protein is OTC, can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any one of SEQ ID NOs:1-6.

[0244] In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, where the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, where the therapeutic protein is Factor VIII, can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any one of SEQ ID NOs:8-14.

[0245] In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, where the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, where the therapeutic protein is Factor IX, can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any one of SEQ ID NOs:15-20.

[0246] In a non-limiting example, an AAV viral vector particle comprising at least one nucleic acid molecule comprising a nucleotide sequence encoding a transposase can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:7.

[0247] In some embodiments of the above method, a composition comprising a nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be administered simultaneously. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be administered sequentially. In some embodiments, a composition comprising a nucleic acid molecule comprising a transposon, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein, and a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding at least one transposase may be administered in close temporal proximity.

[0248] As used herein, the term "proximate in time" refers to administration of one therapeutic composition (e.g., a composition comprising a transposon) occurring within a period of time before or after administration of another therapeutic composition (e.g., a composition comprising a transposase), such that the therapeutic effect of one therapeutic agent overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, the therapeutic effect of one therapeutic agent completely overlaps with the therapeutic effect of the other therapeutic agent. In some embodiments, "proximate in time" means administration of one therapeutic agent occurs within a period of time before or after administration of another therapeutic agent, such that there is a synergistic effect between one therapeutic agent and the other therapeutic agent. "Proximate in time" may vary according to a variety of factors, including, but not limited to, the age, sex, weight, genetic background, medical condition, medical history, and treatment history of the subject to whom the therapeutic agent will be administered, the subject whose disease or condition is being treated or ameliorated, the subject for whom the therapeutic result is being achieved, the dose, frequency, and duration of administration of the therapeutic agent, the pharmacokinetics and pharmacodynamics of the therapeutic agent, and the route by which the therapeutic agent is administered. In some embodiments, "close in time" means within 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, or 8 weeks. In some embodiments, multiple administrations of one therapeutic agent may occur close in time to a single administration of another therapeutic agent. In some embodiments, the close in time may vary during a treatment cycle or dosing regimen.

[0249] In a non-limiting example, the present disclosure provides a method of treating a metabolic liver disorder in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of the LNP of the present disclosure, the LNP comprising at least one DNA molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of the LNP of the present disclosure, the LNP comprising at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the metabolic liver disorder can be ornithine transcarbamylase (OTC) deficiency, and the at least one therapeutic protein can comprise an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, the metabolic liver disorder can be methylmalonic acidemia (MMA), and the at least one therapeutic protein can comprise a methylmalonyl CoA mutase (MUT1) polypeptide.

[0250] In a non-limiting example, the present disclosure provides a method of treating a hemophilia disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of the LNP of the present disclosure, the LNP comprising at least one DNA molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of the LNP of the present disclosure, the LNP comprising at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the hemophilia disease can be hemophilia A and the at least one therapeutic protein can comprise factor VIII. In some embodiments, the hemophilia disease can be hemophilia B and the at least one therapeutic protein can comprise factor IX.

[0251] In a non-limiting example, the present disclosure provides a method for treating metabolic liver disorder in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of AAV viral vector particles comprising at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of LNP of the present disclosure, the LNP comprising at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the metabolic liver disorder can be ornithine transcarbamylase (OTC) deficiency, and the at least one therapeutic protein can comprise an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, the metabolic liver disorder can be methylmalonic acidemia (MMA), and the at least one therapeutic protein can comprise a methylmalonyl CoA mutase (MUT1) polypeptide.

[0252] In a non-limiting example, the present disclosure provides a method of treating hemophilia disease in a subject, the method comprising administering to the subject: a) at least one therapeutically effective amount of an AAV viral vector particle comprising at least one nucleic acid molecule comprising a transposon, the transposon comprising a nucleotide sequence encoding at least one therapeutic protein; and b) at least one therapeutically effective amount of an LNP of the present disclosure, the LNP comprising at least one RNA molecule comprising a nucleotide sequence encoding at least one transposase. In some embodiments, the hemophilia disease can be hemophilia A, and the at least one therapeutic protein can comprise factor VIII. In some embodiments, the hemophilia disease can be hemophilia B, and the at least one therapeutic protein can comprise factor IX.

[0253] The present disclosure provides a method for treating a disease and / or disorder characterized by increased LDL cholesterol, comprising administering to a subject at least one LNP of the present disclosure comprising a genome editing composition, wherein the genome editing composition comprises a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof. In some embodiments, the fusion protein may be a Cas-CLOVER protein. In some embodiments, the genome editing composition may further comprise at least one guide RNA (gRNA) molecule targeting the pcsk9 gene. In some embodiments, the genome editing composition may further comprise at least two gRNA molecules targeting the pcsk9 gene. A gRNA molecule targeting the pcsk9 gene may comprise, consist of, or consist essentially of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs: 29, 30.

[0254] The present disclosure provides a method of reducing LDL cholesterol in a subject in need thereof, the method comprising administering to the subject at least one LNP of the present disclosure comprising a genome editing composition, the genome editing composition comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein, the fusion protein comprising (i) an inactivated Cas9 (dCas9) protein or an inactivated nuclease domain thereof, (ii) a Clo051 protein or a nuclease domain thereof. In some embodiments, the fusion protein can be a Cas-CLOVER protein. In some embodiments, the genome editing composition can further comprise at least one guide RNA (gRNA) molecule targeting the pcsk9 gene. In some embodiments, the genome editing composition can further comprise at least two gRNA molecules targeting the pcsk9 gene. A gRNA molecule targeting the pcsk9 gene may comprise, consist of, or consist essentially of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs: 29, 30.

[0255] In some aspects of the therapeutic methods of the present disclosure, administration of at least one composition and / or nanoparticle of the present disclosure to a subject may result in expression of an exogenous protein (e.g., a therapeutic protein, a transposase, etc.) in at least one organ and / or tissue in the subject.

[0256] In some aspects, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in at least about 10%, or at least about 15%, or at least 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of the cells within the tissue and / or organ.

[0257] In some aspects, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in at least about 10%, or at least about 15%, or at least 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 99% of a specific subpopulation of cells within a tissue and / or organ.

[0258] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in a tissue and / or organ for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0259] In some embodiments, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in specific subpopulations within tissues and / or organs for at least about 1 day, or at least about 2 days, or at least about 3 days, or at least about 4 days, or at least about 5 days, or at least about 6 days, or at least about 7 days, or at least about 8 days, or at least about 9 days, or at least about 10 days.

[0260] In some aspects, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in a tissue and / or organ for about 1 day or less, or about 2 days or less, or about 3 days or less, or about 4 days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less.

[0261] In some aspects, administration of at least one composition and / or nanoparticle of the present disclosure results in expression of the exogenous protein in specific subpopulations within tissues and / or organs for about 1 day or less, or about 2 days or less, or about 3 days or less, or about 4 days or less, or about 5 days or less, or about 6 days or less, or about 7 days or less, or about 8 days or less, or about 9 days or less, or about 10 days or less.

[0262] In some embodiments, the percentage of cells expressing the exogenous protein upon administration of a composition of the present disclosure may be increased by at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, at least 15-fold, or at least about 20-fold, or at least about 25-fold, or at least about 30-fold, or at least about 50-fold compared to the percentage of cells expressing the exogenous protein upon administration of a composition comprising a control nanoparticle.

[0263] In some embodiments, the tissue and / or organ may be the liver. In some embodiments, the specific subpopulation of cells may include, but is not limited to, hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells, or any combination thereof.

[0264] In some embodiments, administration of a composition comprising at least one lipid nanoparticle of the present disclosure is less toxic than administration of a composition comprising at least one control lipid nanoparticle.

[0265] In some embodiments, the reduced toxicity may be manifested as an attenuation of the increase in the level of at least one liver enzyme after administration of at least one lipid nanoparticle of the present disclosure. In some embodiments, the at least one liver enzyme may be one or more of aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP).

[0266] In some embodiments, the reduced toxicity may be manifested as an attenuation of the increase in the level of at least one inflammatory cytokine following administration of at least one lipid nanoparticle of the present disclosure. In some embodiments, the at least one inflammatory cytokine may be one or more of interleukin-6 (IL-6), interferon gamma (INF-G), and tumor necrosis factor alpha (TNF-a).

[0267] In some embodiments, reduced toxicity may be manifested as attenuated weight loss following administration of at least one lipid nanoparticle of the present disclosure.

[0268] In some embodiments, the control lipid nanoparticle is an otherwise identical lipid nanoparticle, except that the cationic lipid is not a bioreducible ionizable cationic lipid.

[0269] In some embodiments, the control lipid nanoparticle is a lipid nanoparticle that does not contain a bioreducible ionizable cationic lipid.

[0270] In some embodiments, the control lipid nanoparticle is a lipid nanoparticle that does not contain ssPalmO-Ph-P4C2.

[0271] In some embodiments, the control lipid nanoparticle is an otherwise identical lipid nanoparticle except that the cationic lipid is not ssPalmO-Ph-P4C2.

[0272] In some embodiments, the control lipid nanoparticle is a lipid nanoparticle that comprises a greater amount of a bioreducible, ionizable cationic lipid.

[0273] In some embodiments, the control lipid nanoparticle is a lipid nanoparticle that comprises a lower amount of a bioreducible ionizable cationic lipid.

[0274] In some aspects, the control lipid nanoparticles comprise lipid nanoparticle compositions already disclosed in the art.

[0275] In some aspects, the control lipid nanoparticles are administered to the subject at the same dose as the lipid nanoparticles of the present disclosure.

[0276] In some aspects, the lipid nanoparticles of the present disclosure can be produced using a microfluidic mixing platform. In some aspects, the microfluidic mixing platform can be a non-turbulent microfluidic mixing platform.

[0277] In some embodiments, the microfluidic mixing platform may use a microfluidic device to combine a miscible solvent phase containing the lipid components of the nanoparticles with an aqueous phase containing the lipid nanoparticle cargo (e.g., nucleic acid, DNA, mRNA, etc.) to produce the lipid nanoparticles of the present disclosure. In some embodiments, the miscible solvent phase and the aqueous phase are mixed in the microfluidic device under laminar flow conditions that do not allow for immediate mixing of the two phases. When the two phases move under laminar flow in a microfluidic channel, the microscopic features of the channel may allow for controlled homogeneous mixing to produce the lipid nanoparticles of the present disclosure.

[0278] In some embodiments, the microfluidic mixing platform may include, but is not limited to, NanoAssemblr® Spark (Precision NanoSystems), NanoAssemblr® Ignite™ (Precision NanoSystems), NanoAssemblr® Benchtop (Precision NanoSystems), NanoAssemblr® Blaze (Precision NanoSystems), or NanoAssemblr® GMP System (Precision NanoSystems).

[0279] In some aspects, the lipid nanoparticles of the present disclosure may be produced using a microfluidic mixing platform that mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0280] In some aspects, the lipid nanoparticles of the present disclosure may be produced using a T-mixer, which mixes at a rate of at least about 2.5 ml / min, or at least about 5 ml / min, or at least about 7.5 ml / min, or at least about 10 ml / min, or at least about 12.5 ml / min, or at least about 15 ml / min, or at least about 17.5 ml / min, or at least about 20 ml / min, or at least about 22.5 ml / min, or at least about 25 ml / min, or at least about 27.5 ml / min, or at least about 30 ml / min.

[0281] In some aspects, the lipid nanoparticles of the present disclosure may be produced using a microfluidic mixing platform that mixes a miscible solvent phase with an aqueous phase in a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solvent:water, v:v.

[0282] In some aspects, lipid nanoparticles of the present disclosure may be produced using a T-mixer, which mixes a miscible solvent phase with an aqueous phase in a ratio of about 10:1, or about 9:1, or about 8:1, or about 7:1, or about 6:1, or about 5:1, or about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4, or about 1:5, or about 1:6, or about 1:7, or about 1:8, or about 1:9, or about 1:10, solvent:water, v:v.

[0283] piggyBac ITR sequence In some aspects, the nucleic acid molecule can comprise a piggyBac ITR sequence. In some aspects, the nucleic acid molecule can comprise a first piggyBac ITR sequence and a second piggyBac ITR sequence.

[0284] In some embodiments, the piggyBac ITR sequence can include any piggyBac ITR sequence known in the art.

[0285] In some embodiments of the methods of the present disclosure, the piggyBac ITR sequence, such as the first piggyBac ITR sequence and / or the second piggyBac ITR sequence, can comprise, consist essentially of, or consist of Sleeping Beauty transposon ITR, Helraiser transposon ITR, Tol2 transposon ITR, TcBuster transposon ITR, or any combination thereof.

[0286] Insulator array In some embodiments, the insulator sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any of the sequences set forth in SEQ ID NOs: 146, 147.

[0287] Promoter sequence In some aspects, the nucleic acid molecule may comprise a promoter sequence. In some aspects, the promoter sequence may comprise any promoter sequence known in the art. In some aspects, the promoter sequence may comprise any liver-specific promoter sequence known in the art.

[0288] In some embodiments, the promoter sequence may comprise a hybrid liver promoter (HLP). In some embodiments, the promoter sequence may comprise an LP1 promoter. In some embodiments, the promoter sequence may comprise a leukocyte specific expression of pp52 (LSP1) long promoter. In some embodiments, the promoter sequence may comprise a thyroxine binding globulin (TBG) promoter. In some embodiments, the promoter sequence may comprise a wTBG promoter. In some embodiments, the promoter sequence may comprise a hepatic combinatorial bundle (HCB) promoter. In some embodiments, the promoter sequence may comprise a 2xApoE-hAAT promoter. In some embodiments, the promoter sequence may comprise a leukocyte-specific expression of pp52 (LSP1) plus chimeric intron promoter. In some embodiments, the promoter sequence may comprise a cytomegalovirus (CMV) promoter.

[0289] Transgene sequence In some embodiments, the transgene sequence can comprise, consist essentially of, or consist of a nucleic acid sequence encoding a methylmalonyl-CoA mutase (MUT1) polypeptide. In some embodiments, the nucleic acid sequence encoding the MUT1 polypeptide can comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NOs: 40-51.

[0290] In some embodiments, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, the nucleic acid sequence encoding a MUT1 polypeptide may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NOs: 56-59.

[0291] In some embodiments, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding an iCAS9 polypeptide.

[0292] In some embodiments, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a factor VIII (FVIII) polypeptide, hi some embodiments, the FVIII polypeptide may be a FVIII polypeptide lacking a B domain (hereinafter referred to as a FVIII-BDD polypeptide).

[0293] In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding a FVIII-BDD polypeptide. In some embodiments, the transgene sequence may comprise a nucleic acid sequence encoding a FVIII-BDD polypeptide, which comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO: 21. In some embodiments, the nucleic acid sequence encoding a FVIII-BDD polypeptide may comprise, consists essentially of, or consists of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any of the sequences set forth in SEQ ID NO: 22.

[0294] In some embodiments, the transgene sequence may comprise, consist essentially of, or consist of a nucleic acid sequence encoding a factor IX (FIX) polypeptide. In some embodiments, the FIX polypeptide may comprise a R338L mutation. As will be appreciated by those skilled in the art, the R338L mutation may be referred to as a Padua mutation. In some embodiments, the nucleic acid sequence encoding the FIX polypeptide may comprise, consist essentially of, or consist of at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 65% of the nucleic acid sequence.

[0295] In some aspects, the transgene sequence may be codon optimized according to methods known in the art.

[0296] In some embodiments, at least one transgene sequence may be operably linked to at least one promoter sequence present in the same polynucleotide.

[0297] Therapeutic Proteins In some embodiments, a therapeutic protein can comprise, consist essentially of, or consist of a methylmalonyl-CoA mutase (MUT1) polypeptide. In some embodiments, the MUT1 polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any one of SEQ ID NOs: 52-55.

[0298] In some embodiments, a therapeutic protein can comprise, consist essentially of, or consist of an ornithine transcarbamylase (OTC) polypeptide. In some embodiments, an OTC polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NOs:60-63.

[0299] In some embodiments, the therapeutic protein may comprise, consist essentially of, or consist of a factor VIII (FVIII) polypeptide, hi some embodiments, the FVIII polypeptide may be a FVIII polypeptide lacking the B domain (hereinafter referred to as a FVIII-BDD polypeptide).

[0300] In some embodiments, the therapeutic protein may comprise, consist essentially of, or consist of a factor IX (FIX) polypeptide. In some embodiments, the FIX polypeptide may comprise a R338L mutation. As will be understood by those skilled in the art, the R338L mutation may be referred to as a Padua mutation. In some embodiments, the FIX polypeptide comprises, consists essentially of, or consists of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to any one of SEQ ID NO:64.

[0301] 3'UTR sequence In some embodiments, the 3'UTR sequence may be an AES 3'UTR sequence, which may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to SEQ ID NO:25.

[0302] In some embodiments, the 3'UTR sequence may be an mtRNR1 3'UTR sequence, which may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:26.

[0303] In some embodiments, the 3'UTR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to SEQ ID NO:27.

[0304] PolyA sequence In some embodiments, the nucleic acid molecule can include a polyA sequence. In some embodiments, the polyA sequence can include any polyA sequence known in the art.

[0305] Self-cleaving peptide sequence In some embodiments, the nucleic acid molecule may comprise a self-cleaving peptide sequence. In some embodiments, the self-cleaving peptide sequence may comprise any self-cleaving peptide sequence known in the art. In some embodiments, the self-cleaving peptide sequence may comprise a 2A self-cleaving peptide sequence known in the art. Non-limiting examples of self-cleaving peptides include T2A peptide, GSG-T2A peptide, E2A peptide, GSG-E2A peptide, F2A peptide, GSG-F2A peptide, P2A peptide, or GSG-P2A peptide.

[0306] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a T2A peptide.

[0307] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-T2A peptide.

[0308] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding an E2A peptide.

[0309] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-E2A peptide.

[0310] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding an F2A peptide.

[0311] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-F2A peptide.

[0312] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a P2A peptide.

[0313] In some embodiments, the self-cleaving peptide sequence may comprise a nucleic acid sequence encoding a GSG-P2A peptide.

[0314] Transposition System In some aspects, the nucleic acid molecule may comprise a transposon or nanotransposon comprising a first nucleic acid sequence, the first nucleic acid sequence comprising (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR, (b) a second ITR or a sequence encoding the second ITR, and (c) an intra-ITR sequence or a sequence encoding the intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding the transposon.

[0315] In some aspects, the nucleic acid molecule may comprise a transposon or nanotransposon comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR) or a sequence encoding the first ITR; (b) a second ITR or a sequence encoding the second ITR; and (c) an intra-ITR sequence or a sequence encoding the intra-ITR, wherein the intra-ITR sequence comprises a transposon sequence or a sequence encoding a transposon; and a second nucleic acid sequence comprising an inter-ITR sequence or a sequence encoding the inter-ITR, wherein the length of the inter-ITR sequence is 700 nucleotides or less.

[0316] The transposon or nanotransposon of the present disclosure comprises a nucleotide sequence encoding a therapeutic protein. The transposon or nanotransposon may be a plasmid DNA transposon comprising a nucleotide sequence encoding a therapeutic protein flanked by two cis-regulatory insulator elements. The transposon or nanotransposon may further comprise a plasmid comprising a sequence encoding a transposase. The sequence encoding the transposase may be a DNA sequence or an RNA sequence. Preferably, the sequence encoding the transposase is an mRNA sequence.

[0317] The transposon or nanotransposon of the present disclosure can be a piggyBac™ (PB) transposon. In some embodiments, when the transposon is a PB transposon, the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, or a Super piggyBac™ (SPB or sPB) transposase. Preferably, the sequence encoding the SPB transposase is an mRNA sequence.

[0318] PB transposons, and non-limiting examples of PB, PBL, and SPB transposases, are described in detail in U.S. Patent No. 6,218,182, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and PCT Publication No. 2010 / 099296.

[0319] The PB, PBL, and SPB transposases recognize transposon-specific inverted terminal repeats (ITRs) at the ends of the transposon and insert their contents between the ITRs at the sequence 5'-TTAT-3' (TTAT target sequence) within the chromosomal site, or at the sequence 5'-TTAA-3' (TTAA target sequence) within the chromosomal site. The target sequences of the PB or PBL transposons are 5'-CTAA-3', 5'-TTAG-3', 5'-ATAA-3', 5'-TCAA-3', 5'AGTT-3', 5'-ATTA-3', 5'-GTTA-3', 5'-TTGA-3', 5'-TTTA-3', 5'-TTAC-3', 5'-ACTA-3', 5'-AGGG-3', 5'-CTAG-3', 5'-TGAA-3', 5'-AGGT-3', 5'-ATCA-3', 5'-CTCC-3', 5'-TAAA-3', 5'-TCTC-3', 5'TGAA-3', 5'-A AAT-3', 5'-AATC-3', 5'-ACAA-3', 5'-ACAT-3', 5'-ACTC-3', 5'-AGTG-3', 5'-ATAG-3', 5'-CAAA-3', 5'-CACA-3', 5'-CATA-3', 5'-CCAG-3', 5'-CCCA-3', 5'-CGTA-3', 5'-GTCC-3', 5'-TAAG-3', 5'-TCTA-3', 5'-TGAG-3', 5'-TGTT-3', 5'-TTCA-3', 5'-TTCT-3', and 5'-TTTT-3'. The PB or PBL transposon system has no payload limit for the gene of interest that can be included between the ITRs.

[0320] Exemplary amino acid sequences of one or more of the PB, PBL, and SPB transposases are disclosed in US Pat. No. 6,218,185, US Pat. No. 6,962,810, and US Pat. No. 8,399,643.

[0321] The PB or PBL transposase may comprise or consist of an amino acid sequence having two or more or three or more amino acid substitutions at each of positions 30, 165, 282, or 538. The transposase may be an SPB transposase comprising or consisting of an amino acid sequence, where the amino acid substitution at position 30 may be a substitution of valine (V) for isoleucine (I), the amino acid substitution at position 165 may be a substitution of serine (S) for glycine (G), the amino acid substitution at position 282 may be a substitution of valine (V) for methionine (M), and the amino acid substitution at position 538 may be a substitution of lysine (K) for asparagine (N).

[0322] In certain embodiments, the transposase comprises the above mutations at positions 30, 165, 282, and / or 538, and the following mutations are also present: , 319, 327, 328, 340, 421, 436, 456, 470, 486, 503, 552, 570, and 591, are described in more detail in PCT Publication Nos. 2019 / 173636 and PCT / US2019 / 049816.

[0323] The PB, PBL, or SPB transposase may be isolated or derived from an insect, vertebrate, crustacean, or urochordate, as described in more detail in PCT Publication Nos. 2019 / 173636 and PCT / US2019 / 049816. In a preferred embodiment, the PB, PBL, or SPB transposase is isolated or derived from the insects Trichoplusia ni (GenBank Accession No. AAA87375) or Bombyx mori (GenBank Accession No. BAD11135).

[0324] A hyperactive PB or PBL transposase is a transposase that is more active than the naturally occurring variant from which it is derived. In a preferred embodiment, the hyperactive PB or PBL transposase is isolated or derived from Bombyx mori or Xenopus tropicalis. Examples of hyperactive PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636. A list of hyperactive amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0325] In some embodiments, the PB or PBL transposase is integration-deficient. An integration-deficient PB or PBL transposase is a transposase that can excise its corresponding transposon, but can integrate the excised transposon at a lower frequency than the corresponding wild-type transposase. Examples of integration-deficient PB or PBL transposases are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636. A list of integration-deficient amino acid substitutions is disclosed in U.S. Patent No. 10,041,077.

[0326] In some embodiments, the PB or PBL transposase is fused to a nuclear localization signal. Examples of PB or PBL transposase fused to a nuclear localization signal are disclosed in U.S. Patent No. 6,218,185, U.S. Patent No. 6,962,810, U.S. Patent No. 8,399,643, and WO2019 / 173636.

[0327] In some embodiments, the sPB protein may comprise, consist essentially of, or consist of an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage therebetween) to any one of SEQ ID NOs:37-39.

[0328] The transposon or nanotransposon of the present disclosure can be a Sleeping Beauty transposon. In some embodiments, when the transposon is a Sleeping Beauty transposon, the transposase is a Sleeping Beauty transposase (e.g., as disclosed in U.S. Pat. No. 9,228,180) or a hyperactive Sleeping Beauty (SB100X) transposase.

[0329] The transposon or nanotransposon of the present disclosure can be a Helraiser transposon. Exemplary Helraiser transposons include Helibat1. In some embodiments, when the transposon is a Helraiser transposon, the transposase is a Helitron transposase (e.g., as disclosed in WO2019 / 173636).

[0330] The transposon or nanotransposon of the present disclosure can be a Tol2 transposon. In some embodiments, when the transposon is a Tol2 transposon, the transposase is a Tol2 transposase (e.g., as disclosed in WO2019 / 173636).

[0331] The transposon or nanotransposon of the present disclosure can be a TcBuster transposon. In some embodiments, when the transposon is a TcBuster transposon, the transposase is a TcBuster transposase or a hyperactive TcBuster transposase (e.g., as disclosed in WO2019 / 173636). The TcBuster transposase can comprise or consist of a naturally occurring amino acid sequence or a non-naturally occurring amino acid sequence. The polynucleotide encoding the TcBuster transposase can comprise or consist of a naturally occurring nucleic acid sequence or a non-naturally occurring nucleic acid sequence.

[0332] In some aspects, the mutant TcBuster transposase comprises one or more sequence variations when compared to a wild-type TcBuster transposase, as described in more detail in PCT Publication Nos. 2019 / 173636 and PCT / US2019 / 049816.

[0333] The cell delivery compositions (e.g., transposons) disclosed herein can include nucleic acid molecules encoding therapeutic proteins or therapeutic agents. Examples of therapeutic proteins include those disclosed in PCT Publication Nos. 2019 / 173636 and PCT / US2019 / 049816.

[0334] Cells and modified cells of the present disclosure The cells and modified cells of the present disclosure may be mammalian cells. Preferably, the cells and modified cells are human cells. In one aspect, the cells targeted for modification using the LNP compositions of the present disclosure are hepatocytes, hepatic stellate cells, Kupffer cells, or hepatic sinusoidal endothelial cells. In one embodiment, the LNP composition comprises at least one mRNA molecule encoding a transposase, and the modified cells are generated in vivo. In one embodiment, the LNP composition comprises at least one DNA molecule encoding a transposon, and the modified cells are generated in vivo. In one embodiment, the transposon comprises a nucleotide sequence encoding a therapeutic gene operably linked to a liver-specific promoter.

[0335] The cells and modified cells of the present disclosure may be somatic cells. The cells and modified cells of the present disclosure may be differentiated cells. The cells and modified cells of the present disclosure may be autologous or allogeneic cells. Allogeneic cells are engineered to prevent adverse reactions to engraftment after administration to a subject. Allogeneic cells may be any type of cell. Allogeneic cells may be stem cells or may be derived from stem cells. Allogeneic cells may be differentiated somatic cells.

[0336] Formulations, Dosages, and Modes of Administration The present disclosure provides formulations, dosages, and methods for administration of the compositions described herein.

[0337] The disclosed compositions and pharmaceutical compositions may further comprise at least one of any suitable auxiliary agent, including, but not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc. Pharmaceutically acceptable auxiliary agents are preferred. Non-limiting examples and preparation methods of such sterile solutions are well known in the art, but are not limited to, for example, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990, and "Physician's Desk Reference", 52nd ed., Medical Economics (Montvale, NJ) 1998. Pharmaceutically acceptable carriers that are suitable for the mode of administration, solubility, and / or stability of the composition, as known in the art or described herein, can be routinely selected.

[0338] For example, the disclosed LNP compositions of the present disclosure can further comprise a diluent. In some compositions, the diluent can be phosphate buffered saline (PBS). In some compositions, the diluent can be sodium acetate.

[0339] Non-limiting examples of pharmaceutical excipients and additives suitable for use include proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, di-, tri-, tetra-, and oligosaccharides, derivatized sugars such as alditols, aldonic acids, esterified sugars, and polysaccharides or sugar polymers), which may be present alone or in combination, including alone or in combinations of 1-99.99% by weight or volume. Non-limiting examples of protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acids / protein components that may also function in a buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, asparagine, and the like. One preferred amino acid is glycine.

[0340] The composition may also include a buffer or pH adjuster, and typically the buffer is a salt prepared from an organic acid or base.Representative buffers include organic acid salts such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid salts, Tris, tromethamine hydrochloride, or phosphate buffers.Preferred buffers are organic acid salts such as citrate.

[0341] Many known and developed modes can be used to administer a therapeutically effective amount of the compositions or pharmaceutical compositions disclosed herein. Non-limiting examples of administration modes include bolus administration, buccal, injection, intra-articular, intra-bronchial, intra-peritoneal, intra-capsular, intra-chondral, intra-cavity, intracellular, intra-cerebellar, intra-ventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intra-lesional, intra-muscular, intra-myocardial, intra-nasal, intra-ocular, intra-osseous, intra-periosteal, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-uterine, intra-tumor, intravenous, intravesical, oral, parenteral, intra-rectal, sublingual, subcutaneous, transdermal, or intra-vaginal means.

[0342] The compositions of the present disclosure may be for use parenterally (subcutaneously, intramuscularly, or intravenously), or for any other administration, particularly in the form of a liquid solution or suspension, for use in vaginal or rectal administration, particularly in semi-solid forms such as, but not limited to, creams and suppositories, for buccal or sublingual administration, including but not limited to, in the form of tablets or capsules, or intranasally, including but not limited to, powders, nasal drops, or aerosols, or certain medications, or transdermally, including but not limited to, gels, ointments, lotions, suspensions, or patch delivery systems that use chemical enhancers such as dimethylsulfoxide to modify the skin structure or increase the drug concentration in transdermal patches (Junginger, et al. In "Drug Permeation Enhancement;" Hsieh, DS, Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994), or the application of electric fields to create temporary transport pathways, such as electroporation, to increase the mobility of charged drugs through the skin, such as iontophoresis, or the application of ultrasound, such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entireties).

[0343] For parenteral administration, any composition disclosed herein may be formulated as a solution, suspension, emulsion, particle, powder, or lyophilized powder in association with or provided separately from a pharma- ceutically acceptable parenteral vehicle. Formulations for parenteral administration may contain, as common excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Aqueous or oily suspensions for injection may be prepared using appropriate emulsifiers or wetting agents and suspending agents according to known methods. Medicaments for injection may be non-toxic, parenterally administrable diluents such as aqueous solutions, sterile injectable solutions, or suspensions in solvents. As usable vehicles or solvents, water, Ringer's solution, isotonic saline, and the like are acceptable, and as ordinary solvents or suspension solvents, sterile non-volatile oils may be used. For these purposes, any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic fatty acids or fatty acids, natural or synthetic or semi-synthetic mono- or di- or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means as described in U.S. Pat. No. 5,851,198, gas pressurized needleless injection devices, and laser perforation devices as described in U.S. Pat. No. 5,839,446.

[0344] For pulmonary administration, preferably, the compositions or pharmaceutical compositions described herein are delivered in a particle size effective to reach the lower airways of the lungs or sinuses. The compositions or pharmaceutical compositions may be delivered by any of a variety of inhalation or nasal devices known in the art for administration of therapeutic agents by inhalation. These devices capable of depositing an aerosolized formulation in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers (e.g., jet nebulizers, ultrasonic nebulizers), dry powder generators, nebulizers, and the like. All such devices may use formulations suitable for administration for dispensing the compositions or pharmaceutical compositions described herein in an aerosol. Such aerosols may consist of either solutions (both aqueous and non-aqueous) or solid particles. Additionally, a spray comprising the compositions or pharmaceutical compositions described herein may be generated by forcing a suspension or solution of at least one protein scaffold through a nozzle under pressure. In a metered dose inhaler (MDI), the propellant, compositions, or pharmaceutical compositions described herein, as well as any excipients or other additives, are contained within a canister as a mixture with a liquefied compressed gas. Actuation of the metered dose valve releases the mixture as an aerosol. A more detailed description of pulmonary administration, formulations, and associated devices is disclosed in PCT Publication No. 2019 / 049816.

[0345] For absorption through mucosal surfaces, the composition includes an emulsion containing a plurality of submicron particles, a viscopolymer, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through the mucosal surface by achieving mucoadhesion of the emulsion particles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application of the emulsions of the present disclosure may include corneal, conjunctival, oral, sublingual, nasal, vaginal, pulmonary, gastric, intestinal, and rectal routes of administration. Formulations for vaginal or rectal administration, such as suppositories, may contain excipients such as polyalkylene glycols, petrolatum, cocoa butter, and the like. Formulations for intranasal administration may be solid, may contain excipients such as lactose, or may be aqueous or oily solutions of nasal drops. For oral administration, excipients include sugars, calcium stearate, magnesium stearate, pregelinized starch, and the like (U.S. Pat. No. 5,849,695). A more detailed description of mucosal administration and formulations is disclosed in PCT Publication No. 2019 / 049816.

[0346] For transdermal administration, the compositions or pharmaceutical compositions disclosed herein are encapsulated in a delivery device, such as liposomes or polymeric nanoparticles, microparticles, microcapsules, or microspheres (collectively referred to as microparticles unless otherwise stated). Some suitable devices are known, including microparticles made from synthetic polymers, such as polyhydroxy acids, such as polylactic acid, polyglycolic acid, and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers, such as collagen, polyamino acids, albumin, and other proteins, alginates, and other polysaccharides, and combinations thereof (U.S. Patent No. 5,814,599). A more detailed description of transdermal administration, formulations, and suitable devices is disclosed in PCT Publication No. 2019 / 049816.

[0347] It may be desirable to deliver the disclosed compounds to a subject for extended periods of time, for example, from one week to one year from a single administration. A variety of sustained release, depot, or implant dosage forms may be utilized.

[0348] Suitable dosages are well known in the art.See, for example, Wells et al., eds., Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook of Drugs, 21st edition, Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ. A preferred dose can optionally include about 0.1-99 and / or 100-500 mg / kg / dose, or any range, value, or fraction thereof, or to achieve a serum concentration of about 0.1-5000 μg / ml, or any range, value, or fraction thereof, per single or multiple doses. A preferred dose range for the compositions or pharmaceutical compositions disclosed herein is about 1 mg / kg, up to about 3, about 6, or about 12 mg / kg of subject body weight.

[0349] Alternatively, the dose administered may vary depending on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration, the age, health, and weight of the recipient, the nature and extent of the symptoms, type of concurrent therapy, frequency of treatment, and the desired effect.

[0350] As a non-limiting example, human or animal treatment can be provided as a single or periodic dose of a composition or pharmaceutical composition disclosed herein, about 0.1 to 100 mg / kg, or any range, value, or fraction thereof, per day, for at least one day of days 1 to 40, or alternatively or additionally, for at least one week of weeks 1 to 52, or alternatively or additionally, for at least one year of years 1 to 20, or any combination thereof.

[0351] As disclosed herein, in embodiments in which the composition administered to a subject in need thereof is a modified cell, the cells may be in the form of a modified cell, such as a modified cell line comprising about 1×10 3 ~1×10 15 cells, 1 x 10 3 ~1×10 15 cells, approximately 1 x 10 4 ~1×10 12 cells, approximately 1 x 10 5 ~1×10 10 cells, approximately 1 x 10 6 ~1×10 9 cells, approximately 1 x 10 6 ~1×10 8 cells, approximately 1 x 10 6 ~1×10 7 cells, or approximately 1 x 10 6 ~25×10 6 In one embodiment, the cells may be administered at about 5×10 6 ~25×10 6 Each patient is administered one dose.

[0352] A more detailed description of the pharma- ceutically acceptable excipients, formulations, dosages, and methods of administration of the compositions and pharmaceutical compositions of the present disclosure are disclosed in PCT Publication No. 2019 / 04981.

[0353] The present disclosure provides for the use of the disclosed compositions and pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject, as known in the art or as described herein, e.g., by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one aspect, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0354] Any use or method of the present disclosure may comprise administering an effective amount of any composition or pharmaceutical composition disclosed herein to a cell, tissue, organ, animal, or subject in need of such modification, treatment, or therapy. Such methods optionally further comprise co-administration or combination therapy for treating such disease or disorder, and administration of any composition or pharmaceutical composition disclosed herein further comprises administration prior to, concomitantly with, and / or following at least one chemotherapeutic agent (e.g., alkylating agent, mitotic inhibitor, radiopharmaceutical).

[0355] In some aspects, the subject does not develop graft versus host (GvH) and / or host versus graft (HvG) after administration. In one aspect, administration is systemic. Systemic administration can be by any means known in the art and described in detail herein. Preferably, systemic administration is by intravenous injection or infusion. In one aspect, administration is local. Local administration can be by any means known in the art and described in detail herein. Preferably, local administration is by intratumoral, intraspinal, intraventricular, intraocular, or intraosseous injection or infusion.

[0356] In some embodiments, the therapeutically effective dose is a single dose. In some embodiments, the single dose is at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or any number of doses during simultaneous production. In some embodiments where the composition is autologous or allogeneic cells, the dose is sufficient for the cells to engraft and / or persist for a sufficient time to treat a disease or disorder.

[0357] In some aspects of the methods of treatment described herein, the treatment may be modified or terminated. Specifically, in aspects in which the composition used in the treatment comprises an inducible pro-apoptotic polypeptide, apoptosis may be selectively induced in the cells by contacting the cells with an inducer. The treatment may be modified or terminated, for example, in response to signs of recovery or signs of reduced disease severity / progression, signs of disease remission / failure, and / or the occurrence of an adverse event. In some aspects, the method includes administering an inhibitor of the inducer to inhibit modification of the cell therapy, thereby restoring the function and / or efficacy of the cell therapy (e.g., when signs or symptoms of disease recurrence or increased severity, and / or adverse events are resolved).

[0358] nucleic acid molecule The nucleic acid molecules of the present disclosure that code for a therapeutic protein can be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning or produced synthetically, or any combination thereof. The DNA can be triple-stranded, double-stranded, or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or the non-coding strand, also called the antisense strand.

[0359] The isolated nucleic acid molecules of the present disclosure may include nucleic acid molecules that include one or more introns, for example, but not limited to, an open reading frame (ORF), optionally including at least one specific enzymatically active portion of a therapeutic protein, and nucleic acid molecules that include a coding sequence for a therapeutic protein, as well as nucleic acid molecules that are substantially different from those described above, but that still code for a therapeutic protein due to the degeneracy of the genetic code, as described herein and / or known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for a person skilled in the art to generate such degenerate nucleic acid variants that code for a particular protein scaffold of the present disclosure. For example, see Ausubel et al., supra, such nucleic acid variants are included in the present disclosure.

[0360] As provided herein, the nucleic acid molecules of the present disclosure, including those encoding a Therapeutic protein, may include additional coding sequences that code for additional amino acids, such as those that provide additional functions, such as coding sequences for at least one signal leader or fusion peptide, with or without additional coding sequences as described above, such as at least one intron, together with additional non-coding sequences, including, but not limited to, non-coding 5' and 3' sequences that by themselves code for the amino acid sequence of an enzymatically active fragment of a Therapeutic protein, coding sequences for the entire Therapeutic protein or a portion thereof, transcribed non-translated sequences that play a role in mRNA processing, including splicing and polyadenylation signals (e.g., ribosome binding and mRNA stability). Thus, the coding sequence for a Therapeutic protein may be fused to a marker sequence, such as a sequence that codes for a peptide that facilitates purification of the fused Therapeutic protein.

[0361] Nucleic acid construction Isolated nucleic acids of the disclosure can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and / or (d) combinations thereof, which are well known in the art.

[0362] The nucleic acid may conveniently include sequences in addition to the polynucleotide of the present disclosure. For example, a multiple cloning site containing one or more endonuclease restriction sites may be inserted into the nucleic acid to aid in the isolation of the polynucleotide. Also, a translatable sequence may be inserted to aid in the isolation of the translated polynucleotide of the present disclosure. For example, a hexahistidine marker sequence provides a convenient means for purifying the protein of the present disclosure. The nucleic acid of the present disclosure, except for the coding sequence, is optionally a vector, adapter, or linker for cloning and / or expressing the polynucleotide of the present disclosure.

[0363] Additional sequences may be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides, or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra, or Sambrook, supra).

[0364] Recombinant methods for constructing nucleic acids The isolated nucleic acid compositions of the present disclosure, such as RNA, cDNA, genomic DNA, or any combination thereof, can be obtained from biological sources using any number of cloning methodologies known to those skilled in the art. In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the present disclosure under stringent conditions are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art. (See, for example, Ausubel, supra, or Sambrook, supra).

[0365] Nucleic Acid Screening and Isolation Methods A cDNA or genomic library may be screened using a probe based on the sequence of the polynucleotide of the present disclosure. The probe may be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those skilled in the art will appreciate that various degrees of stringency of hybridization may be employed in the assay, and either the hybridization or the wash medium may be stringent. The more stringent the conditions for hybridization, the higher the degree of complementarity between the probe and the target must be for duplex formation to occur. The degree of stringency may be controlled by one or more of temperature, ionic strength, pH, and the presence of a partially denaturing solvent such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution, for example, through manipulation of the concentration of formamide in the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary depending on the stringency of the hybridization medium and / or the wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein, however, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and / or wash medium.

[0366] Methods for amplification of RNA or DNA are well known in the art and can be used in accordance with the present disclosure without undue experimentation, based on the teaching and guidance provided herein.

[0367] Known methods of DNA or RNA amplification include, but are not limited to, the polymerase chain reaction (PCR) and related amplification processes (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and 4,965,188 to Mullis et al., U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor et al., U.S. Pat. No. 5,142,033 to Innis, U.S. Pat. No. 5,122,464 to Wilson et al., U.S. Pat. No. 5,091,310 ... Innis, U.S. Pat. No. 5,091,310 to Gyllensteiger et al., U.S. Pat. No. 5,122,464 to Wilson et al., U.S. Pat. No. 5,091,310 to Innis, U.S. Pat. No. 5,122,464 to Gyllensteiger et al., U.S. Pat. No. 5,122,464 to Gyllensteiger et al., U.S. Pat. No. 5,091,310 to Gyllensteiger, U.S. Pat. No. 5,122,464 to Gyllensteiger, U.S. Pat. No. 5,122,464 to Gyllensteiger, U.S. Pat. No. 5,122,464 to Gyllensteiger, U.S. Pat. No. 5,066,584 to N et al., No. 4,889,818 to Gelfand et al., No. 4,994,370 to Silver et al., No. 4,766,067 to Biswas, No. 4,656,134 to Ringold), and RNA-mediated amplification using antisense RNA against a target sequence as a template for double-stranded DNA synthesis (U.S. Patent No. 5,130,238 to Malek et al. under the trade name NASBA), the entire contents of which are incorporated herein by reference. (See, for example, Ausubel, supra, or Sambrook, supra.)

[0368] For example, polymerase chain reaction (PCR) techniques can be used to amplify sequences of the polynucleotides and related genes of the present disclosure directly from genomic DNA or cDNA libraries. PCR and other in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences encoding expressed proteins and generate nucleic acids for use as probes for detecting the presence of desired mRNA in a sample, for nucleic acid sequencing, or for other purposes. Examples of techniques sufficient to guide the skilled artisan through in vitro amplification methods can be found in Berger, Sambrook, and Ausubel, supra, as well as U.S. Patent No. 4,683,202 (1987) to Mullis et al., and Innis, et al., PCR Protocols A Guide to Methods and Applications, Eds., Academic Press Inc., San Diego, Calif. (1990). Commercially available kits for genomic PCR amplification are known in the art. See, for example, Advantage-GC Genomic PCR Kit (Clontech). In addition, for example, T4 gene 32 protein (Boehringer Mannheim) can be used to improve the yield of long PCR products.

[0369] Synthetic methods for constructing nucleic acids The isolated nucleic acid of the present disclosure can also be prepared by direct chemical synthesis according to known methods (see, for example, Ausubel et al., supra). Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence or polymerization with a DNA polymerase using the single strand as a template. Those skilled in the art will recognize that chemical synthesis of DNA can be limited to sequences of about 100 bases or more, but longer sequences can be obtained by ligation of shorter sequences.

[0370] Recombinant Expression Cassettes The present disclosure further provides a recombinant expression cassette comprising the nucleic acid of the present disclosure. The nucleic acid sequence of the present disclosure, for example, a cDNA or a genomic sequence encoding a protein scaffold of the present disclosure, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. The recombinant expression cassette will typically include a polynucleotide of the present disclosure operably linked to a transcription initiation control sequence that directs the transcription of the polynucleotide in the intended host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct the expression of the nucleic acid of the present disclosure.

[0371] In some aspects, isolated nucleic acids functioning as promoters, enhancers, or other elements can be introduced into the appropriate location (upstream, downstream, or intron) of a non-heterologous form of a polynucleotide of the disclosure to up- or down-regulate expression of the polynucleotide of the disclosure. For example, endogenous promoters can be modified in vivo or in vitro by mutation, deletion, and / or substitution.

[0372] Expression vectors and host cells The present disclosure also relates to vectors comprising the isolated nucleic acid molecules of the present disclosure, host cells genetically engineered with the recombinant vectors, and the production of at least one therapeutic protein by recombinant techniques, which are well known in the art (see, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each of which is incorporated herein by reference in its entirety).

[0373] The polynucleotide may be optionally linked to a vector containing a selectable marker for propagation in a host. Generally, the plasmid vector is introduced into a precipitate such as a calcium phosphate precipitate, or into a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into a host cell.

[0374] The DNA insert should be operably linked to a suitable promoter. The expression construct will further contain sites for transcription initiation, termination, and, within the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will preferably include a translation initiation at the beginning and a termination codon (e.g., UAA, UGA, or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG being preferred for mammalian or eukaryotic expression.

[0375] The expression vector will preferably and optionally include at least one selectable marker, such as, but not limited to, ampicillin, zeocin (Sh bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), DHFR (encodes dihydrofolate reductase and confers resistance to methotrexate), mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464, 5,770,359, and 5,827,739), blasticidin (bsd gene), and other selectable markers for eukaryotic cell culture and expression of ... Resistance genes for cytochrome P450 (bla gene), puromycin (pac gene), hygromycin B (hygB gene), G418 / Geneticin (neo gene), kanamycin, spectinomycin, streptomycin, carbenicillin, bleomycin, erythromycin, polymyxin B, or tetracycline resistance genes for culturing in E. coli and other bacteria or prokaryotes (the above patents are incorporated herein by reference in their entirety). Appropriate culture media and conditions for the above host cells are known in the art. Suitable vectors will be readily apparent to those of skill in the art. Introduction of the vector construct into the host cell can be by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection, or other known methods. Such methods are explained in the art, such as Sambrook, supra, chapters 1-4 and 16-18, and Ausubel, supra, chapters 1, 9, 13, 15, 16.

[0376] The expression vector will preferably, and optionally, include at least one selectable cell surface marker for isolation of cells modified by the compositions and methods of the present disclosure. The selectable cell surface markers of the present disclosure include surface proteins, glycoproteins, or proteins that distinguish a cell or a subpopulation of cells from another defined subpopulation of cells. Preferably, the selectable cell surface marker distinguishes cells modified by the compositions or methods of the present disclosure from cells that are not modified by the compositions or methods of the present disclosure. Such cell surface markers include, for example, but are not limited to, "cluster of designation or classification determinant" proteins (often abbreviated as "CD"), such as truncated or full-length forms of CD19, CD271, CD34, CD22, CD20, CD33, CD52, or any combination thereof. The cell surface markers further include the suicide gene marker RQR8 (Philip B et al. Blood. 2014 Aug 21; 124(8): 1277-87).

[0377] The expression vector will preferably, and optionally, include at least one selectable drug resistance marker for isolation of cells modified by the compositions and methods of the present disclosure. The selectable drug resistance markers of the present disclosure may include wild-type or mutant Neo, DHFR, TYMS, FRANCF, RAD51C, GCS, MDR1, ALDH1, NKX2.2, or any combination thereof.

[0378] At least one protein scaffold of the present disclosure may be expressed in modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For example, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the protein scaffold to improve stability and persistence in the host cell during purification or subsequent handling and storage. Also, peptide moieties may be added to the protein scaffold of the present disclosure to facilitate purification. Such regions may be removed prior to final preparation of the protein scaffold or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74, Ausubel, supra, Chapters 16, 17 and 18.

[0379] Those skilled in the art are familiar with the many expression systems available for expressing nucleic acid molecules encoding the proteins of the present disclosure.Alternatively, the nucleic acids of the present disclosure can be expressed in host cells by turning on (by engineering) in the host cells containing endogenous DNA encoding the protein scaffolds of the present disclosure.Such methods are well known in the art, as described, for example, in U.S. Patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, which are fully incorporated herein by reference.

[0380] Exemplary cell cultures useful for the production of protein scaffolds, specific portions or variants thereof are bacterial, yeast, and mammalian cells known in the art. Mammalian cell systems are often in the form of monolayers of cells, although mammalian cell suspensions or bioreactors may also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art and include COS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653, SP2 / 0-Ag14, 293 cells, HeLa cells, and the like, which are readily available, for example, from the American Type Culture Collection, Manassas, Va. (www.atcc.org). Preferred host cells include cells of lymphoid origin, such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession No. CRL-1580) and SP2 / 0-Ag14 cells (ATCC Accession No. CRL-1851). In a preferred embodiment, the recombinant cell is a P3X63Ab8.653 or SP2 / 0-Ag14 cell.

[0381] Expression vectors for these cells can include one or more of the following expression control sequences, including, but are not limited to, an origin of replication, a promoter (e.g., late or early SV40 promoter, CMV promoter (U.S. Pat. Nos. 5,168,062, 5,385,839), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1 alpha promoter (U.S. Pat. No. 5,266,491), at least one human promoter, enhancer, and / or processing information sites such as ribosome binding sites, RNA slicing sites, polyadenylation sites (e.g., SV40 large T Ag polyA addition site), and transcription termination sequences. See, e.g., Ausubel et al., supra; Sambrook et al., supra. Other cells useful for producing the nucleic acids or proteins of the disclosure are known and / or available, e.g., from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.

[0382] When eukaryotic host cells are used, polyadenylation or transcription termination sequences are typically incorporated into the vector. An example of a termination sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences for controlling replication in host cells may be incorporated into the vector, as known in the art.

[0383] Amino acid code The amino acids that make up the protein scaffolds of the present disclosure are often omitted. Amino acid designations may be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon, as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994). Therapeutic proteins of the present disclosure may contain one or more amino acid substitutions, deletions, or additions, either naturally occurring or from mutations and / or human manipulation, as specified herein. Amino acids in the therapeutic proteins of the present disclosure that are essential for function may be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (e.g., Ausubel, supra, Chapter 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to, at least one neutralizing activity. Sites that are important for maintaining activity of a therapeutic protein can also be identified by structural analysis, such as crystallization, nuclear magnetic resonance, or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

[0384] As will be appreciated by those of skill in the art, the present disclosure includes at least one bioactive therapeutic protein of the present disclosure. A bioactive therapeutic protein has a specific activity that is at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95% to 99% or more of the specific activity of a natural (non-synthetic), endogenous, or related and known protein scaffold. Methods for assaying and quantifying the measurement of enzymatic activity and substrate specificity are well known to those of skill in the art.

[0385] In another aspect, the disclosure relates to therapeutic proteins and fragments described herein, which are modified by the covalent attachment of an organic moiety. Such modifications may generate protein scaffold fragments with improved pharmacokinetic properties (e.g., increased in vivo serum half-life). The organic moiety may be a linear or branched hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. In certain aspects, the hydrophilic polymer group may have a molecular weight of about 800 to about 120,000 daltons and may be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), a carbohydrate polymer, an amino acid polymer, or polyvinylpyrrolidone, and the fatty acid or fatty acid ester group may contain about 8 to about 40 carbon atoms.

[0386] The modified therapeutic proteins and fragments of the present disclosure may include one or more organic moieties that are directly or indirectly covalently attached to the antibody. Each organic moiety attached to the protein scaffold or fragment of the present disclosure may be independently a hydrophilic polymer group, a fatty acid group, or a fatty acid ester group. As used herein, the term "fatty acid" includes mono-carboxylic acid and di-carboxylic acid. As used herein, the term "hydrophilic polymer group" refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, therapeutic proteins modified by the covalent attachment of polylysine are encompassed by the present disclosure. Suitable hydrophilic polymers for modifying therapeutic proteins of the present disclosure may be linear or branched and may include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG, etc.), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartic acid, etc.), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), and polyvinylpyrrolidone. Preferably, the hydrophilic polymers for modifying therapeutic proteins of the present disclosure have a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG20,000, where the subscript is the average molecular weight of the polymer in Daltons, may be used. The hydrophilic polymer group may be substituted with one to about six alkyl groups, fatty acid groups, or fatty acid ester groups. Hydrophilic polymers substituted with fatty acid or fatty acid ester groups may be prepared by suitable methods. For example, a polymer containing an amine group can be coupled to a carboxylate of a fatty acid or fatty acid ester, and an activated carboxylate on the fatty acid or fatty acid ester (e.g., activated with N,N-carbonyldiimidazole) can be coupled to a hydroxyl group on the polymer.

[0387] The fatty acids and fatty acid esters suitable for modifying the therapeutic proteins of the present disclosure may be saturated or may contain one or more unsaturated units. Fatty acids suitable for modifying the protein scaffolds of the present disclosure include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristic acid), n-octadecanoate (C18, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), cis-Δ9-octadecanoate (C18, oleate), all cis-Δ5,8,11,14-eicosatetraenoic acid (C20, arachidonic acid), octanedisic acid, tetradecanedisic acid, octadecanedioic acid, docosanedionic acid, and the like. Suitable fatty acid esters include monoesters of dicarboxylic acids containing a linear or branched lower alkyl group. The lower alkyl group can contain from 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

[0388] Modified therapeutic proteins and fragments can be prepared using suitable methods, such as, for example, reaction with one or more modifying agents. The term "modifying agent" as used herein refers to a suitable organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester) that contains an activating group. An "activating group" is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifying agent and the second chemical group. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be attached to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form phosphoramidate or phosphorimide bonds. Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Activating groups can be directly attached to organic groups (e.g., hydrophilic polymers, fatty acids, fatty acid esters) or can be attached via a linker moiety, e.g., a divalent C1-C12 group, where one or more carbon atoms can be replaced by heteroatoms such as oxygen, nitrogen, or sulfur. Suitable linker moieties include, for example, tetraethylene glycol, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH-, and -CH2-O-CH2-CH2-O-CH2-CH2-O-CH-NH-. Modifiers containing a linker moiety can be generated, for example, by reacting a mono-Boc-alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine which can be coupled to another carboxylate as described, or the resulting product can be reacted with maleic anhydride and cyclized to produce an activated maleimide derivative of a fatty acid (see, for example, WO 92 / 16221 to Thompson et al., the teachings of which are incorporated herein by reference in their entirety).

[0389] Modified therapeutic proteins of the disclosure can be generated by reacting a protein scaffold or fragment with a modifying agent. For example, organic moieties can be attached to a protein scaffold in a non-site-specific manner by using an amine-reactive modifying agent, such as an NHS ester of PEG. Modified therapeutic proteins and fragments containing organic moieties attached to specific sites of the protein scaffolds of the present disclosure can be prepared using suitable methods such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1):59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)) and methods described in Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996).

[0390] definition As used throughout this disclosure, the singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes a plurality of such methods, and reference to "a dosage" includes reference to one or more dosages and equivalents known to those skilled in the art.

[0391] "About" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean within one or more standard deviations. Alternatively, "about" can mean a range of a given value of up to 20%, or up to 10%, or up to 5%, or up to 1%. Alternatively, particularly with respect to biological systems or processes, the term can mean a value within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold. When a particular value is described in the present application and claims, unless otherwise stated, the term "about" should be assumed to mean within an acceptable error range for that particular value.

[0392] It will be understood that the compounds disclosed herein may be presented without a specific configuration (e.g., without a specific stereochemistry). Such presentation is intended to encompass all available isomers, tautomers, positional isomers, and stereoisomers of the compound. In some embodiments, presentation of a compound herein without a specific configuration is intended to refer to each of the available isomers, tautomers, positional isomers, and stereoisomers of the compound, or any mixture thereof.

[0393] It should be understood that the compounds described herein include the compounds themselves, as well as their salts and solvates, if applicable. For example, salts can be formed between anions and positively charged groups (e.g., amino) on the substituted compounds disclosed herein. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutaric acid, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).

[0394] The present disclosure provides isolated or substantially purified polynucleotide or protein compositions. An "isolated" or "purified" polynucleotide or protein, or a biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or protein found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or protein is substantially free of other cellular materials or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, a polynucleotide does not contain sequences that naturally flank the polynucleotide in the genomic DNA of the organism from which the polynucleotide is derived (i.e., sequences located at the 5' and 3' ends of the polynucleotide) (optimal protein coding sequences). For example, in various aspects, an isolated polynucleotide may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the polynucleotide in the genomic DNA of the cell from which the polynucleotide is derived. Proteins that are substantially free of cellular material include preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When a protein of the present disclosure or a biologically active portion thereof is recombinantly produced, an optimal culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein chemicals of interest.

[0395] The present disclosure provides fragments and variants of the DNA sequences and proteins of the present disclosure encoded by these DNA sequences. As used throughout the present disclosure, the term "fragment" refers to a portion of a DNA sequence or a portion of an amino acid sequence and thus the protein encoded thereby. A fragment of a DNA sequence, including a coding sequence, may code for a protein fragment that retains the biological activity of the native protein, and thus the DNA recognition or binding activity to a target DNA sequence as described herein. Alternatively, a fragment of a DNA sequence that is useful as a hybridization probe generally does not code for a protein that retains biological activity or does not retain promoter activity. Thus, a fragment of a DNA sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the present disclosure.

[0396] The nucleic acids or proteins of the present disclosure may be constructed by a modular approach, including pre-assembling the monomer units and / or repeat units in a target vector, which may then be assembled into a final destination vector. The polypeptides of the present disclosure may include repeat monomers of the present disclosure, and may be constructed by a modular approach, including pre-assembling the repeat units in a target vector, which may then be assembled into a final destination vector. The present disclosure provides polypeptides produced by the present method, as well as nucleic acid sequences encoding these polypeptides. The present disclosure provides host organisms and cells comprising nucleic acid sequences encoding the polypeptides produced by this modular approach.

[0397] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific.

[0398] The term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements of any essentially significant element to the combination when used for the intended purpose. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants or inert carriers. "Consisting of" means excluding more than trace elements and substantial method steps of other ingredients. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0399] The term "epitope" refers to an antigenic determinant of a polypeptide. An epitope may contain three amino acids that are unique to the epitope in a spatial structure. Generally, an epitope consists of at least 4, 5, 6, or 7 such amino acids, and more usually, at least 8, 9, or 10 such amino acids. Methods for determining the spatial structure of amino acids are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.

[0400] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0401] "Gene expression" refers to the conversion of the information contained within a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, shRNA, microRNA, structural RNA, or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNA that is modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0402] "Modulation" or "regulation" of gene expression refers to a change in the activity of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression.

[0403] The term "operably linked" or its equivalents (e.g., operably linked) means that two or more molecules are positioned relative to each other such that they can interact to affect a function ascribed to one or both of the molecules, or a combination thereof.

[0404] Non-covalently linked components and methods of making and using non-covalently linked components are disclosed. The various components can take a variety of different forms, as described herein. For example, non-covalently linked (i.e., operably linked) proteins can be used to allow for temporary interactions that avoid one or more problems in the art. The ability of non-covalently linked components, such as proteins, to associate and dissociate allows for functional association only or primarily under circumstances where such association is required for the desired activity. The association can be of sufficient duration to allow for the desired effect.

[0405] Methods for targeting a protein to a specific locus in the genome of an organism are disclosed. The methods can include providing a DNA localization component and providing an effector molecule, where the DNA localization component and the effector molecule can be operably linked via a non-covalent bond.

[0406] The term "scFv" refers to a single chain variable fragment. An scFv is a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin connected with a linker peptide. The linker peptide can be about 5-40 amino acids, or about 10-30 amino acids, or about 5, 10, 15, 20, 25, 30, 35, or 40 amino acids in length. Single chain variable fragments lack the constant Fc region found in intact antibody molecules and therefore the consensus binding site (e.g., protein G) used to purify antibodies. The term further includes scFvs that are intracellular antibodies, antibodies that are stable in the cytoplasm of a cell and can bind to intracellular proteins.

[0407] The term "single domain antibody" refers to an antibody fragment having a single monomeric variable antibody domain capable of selectively binding to a specific antigen. Single domain antibodies are generally peptide chains of about 110 amino acids in length that contain one variable domain (VH) of a heavy chain antibody, or of a common IgG, which generally have similar affinity for antigen as whole antibodies, but are more heat-resistant and stable to detergents and high concentrations of urea. Examples are those derived from camelid or fish antibodies. Alternatively, single domain antibodies can be made from common mouse or human IgG with four chains.

[0408] As used herein, the terms "specifical...

Claims

1. A combination pharmaceutical for use in treating at least one disease or disorder in a subject in need thereof, comprising: The combination drug is i) an adeno-associated virus (AAV) viral vector particle comprising at least one nucleic acid molecule comprising a transposon, the viral vector particle, wherein the transposon comprises a nucleotide sequence encoding at least one therapeutic protein; ii) lipid nanoparticles comprising at least one nucleic acid comprising a nucleotide sequence encoding at least one transposase, The lipid nanoparticles comprise at least one cationic lipid, at least one phospholipid, at least one structured lipid, and at least one PEGylated lipid.

2. The method of claim 1, wherein the at least one cationic lipid is ssPalmO-Ph-P4C2; said at least one structured lipid is cholesterol; the at least one phospholipid is dioleoylphosphatidylethanolamine (DOPE); and The pharmaceutical combination for use according to claim 1, wherein said at least one PEGylated lipid is DMG-PEG2000.

3. The lipid nanoparticles, 53 mol% to 60 mol% of ssPalmO-Ph-P4C2; 34 mol% to 41 mol% cholesterol; 4 mol % to 11 mol % of DOPC, DSPC, or DOPE; 0.5 mol % to 2 mol % of DMG-PEG2000; The pharmaceutical combination according to claim 2, wherein the ratio of lipid to nucleic acid in the nanoparticle is from 70:1 to 105:1 (w / w).

4. The lipid nanoparticles, 54 mol% of ssPalmO-Ph-P4C2; 35 mol % cholesterol; 10 mol % DOPC; 1 mol % DMG-PEG2000; wherein the at least one nucleic acid molecule comprises at least one RNA molecule, and The pharmaceutical combination according to claim 3, wherein the ratio of lipid to nucleic acid in the nanoparticle is 100:1 (w / w).

5. The pharmaceutical combination according to claim 4 , wherein the RNA molecule is an mRNA molecule.

6. The pharmaceutical combination according to any one of claims 1 to 5, wherein the transposase is a piggyBac™ (PB) transposase, a piggyBac-like (PBL) transposase, a Super piggyBac™ (SPB) transposase polypeptide, a Sleeping Beauty transposase, a Hyperactive Sleeping Beauty (SB100X) transposase, a Helitron transposase, a Tol2 transposase, a TcBuster transposase, or a mutant TcBuster transposase.

7. The pharmaceutical combination of claim 6, wherein the transposase is a piggyBac™ (PB) transposase or a Super piggyBac™ (SPB) transposase polypeptide.

8. The pharmaceutical combination according to any one of claims 1 to 7, wherein the at least one therapeutic protein is ornithine transcarbamylase (OTC) or methylmalonyl-CoA mutase (MUT1).

9. The combination pharmaceutical of claim 8, wherein the at least one therapeutic protein is an OTC.

10. The combination pharmaceutical described in claim 9, wherein the AAV viral vector particle comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 1 to 6.

11. The combination pharmaceutical of any one of claims 1 to 10, wherein the at least one disease or disorder is a liver disease or liver disorder.

12. The combination pharmaceutical of claim 11, wherein the liver disease or liver disorder is a metabolic liver disorder.

13. The metabolic liver disorder is i) a urea cycle disorder, or ii) The combination medicine according to claim 11 or 12, which is N-acetylglutamate synthase (NAGS) deficiency, carbamoyl phosphate synthase I deficiency (CPSI deficiency), ornithine transcarbamylase (OTC) deficiency, argininosuccinic acid synthase deficiency (ASSD) (citrullinemia I), citrin deficiency (citrullinemia II), argininosuccinic acid lyase deficiency (argininosuccinic aciduria), arginase deficiency (hyperargininemia), ornithine translocase deficiency (HHH syndrome), progressive familial intrahepatic cholestasis type 1 (PFIC1), progressive familial intrahepatic cholestasis type 1 (PFIC2), progressive familial intrahepatic cholestasis type 1 (PFIC3), or any combination thereof.

14. The combination pharmaceutical of any one of claims 1 to 7, wherein the at least one therapeutic protein is factor VIII or factor IX.

15. The method of claim 1, wherein i) the factor IX comprises a R338L mutation; or The pharmaceutical combination according to claim 14, wherein ii) said FVIII protein is a FVIII-BDD polypeptide.

16. The pharmaceutical combination according to claim 13 or 14, wherein the at least one therapeutic protein is factor VIII and the AAV viral vector particle comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 8-14.

17. The pharmaceutical combination according to claim 13 or 14, wherein the at least one therapeutic protein is Factor IX and the AAV viral vector particle comprises a nucleic acid sequence that is at least 95% identical to any one of SEQ ID NOs: 15-20.

18. The pharmaceutical combination according to any one of claims 1 to 7 and 14 to 17, wherein the at least one disease or disorder is a hemophilic disease.

19. The pharmaceutical combination of claim 18, wherein the hemophilic disease is hemophilia A, hemophilia B, hemophilia C, or any combination thereof.

20. The combination pharmaceutical of any one of claims 1 to 19, wherein the AAV viral vector particle and the lipid nanoparticle are administered to the subject in close temporal proximity.

21. The combination pharmaceutical described in any one of claims 1 to 19, wherein the AAV viral vector particles and the lipid nanoparticles are administered to the subject simultaneously.

22. The combination pharmaceutical described in any one of claims 1 to 19, wherein the AAV viral vector particles and the lipid nanoparticles are administered sequentially to the subject.

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