Stealth lipid nanoparticle compositions for cell targeting

Abstract

The present disclosure provides stealth lipid nanoparticle (LNP) compositions engineered to target specific tissues or cell types, e.g., T cells, B cells, and natural killer cells, and genetically modify the cells with therapeutic nucleic acids encapsulated in the LNPs. The present disclosure also provides compositions and methods for making LNPs and treatments using them. LNPs can encapsulate various types of cargo, such as nucleic acids encoding desired therapeutic proteins (e.g., chimeric antigen receptors, enzymes, antibodies, etc.) or nucleic acids with sequences for gene / base editing templates. Nucleic acid molecules can be various forms of double-stranded DNA, single-stranded DNA, or RNA (mRNA, siRNA).

Description

[Technical Field]

[0001] Related Applications This application is a continuation of U.S. Provisional Patent Application No. 63 / 453,616, filed March 21, 2023; U.S. Provisional Patent Application No. 63 / 545,474, filed October 24, 2023; U.S. Provisional Patent Application No. 63 / 429,267, filed December 1, 2022; U.S. Provisional Patent Application No. 63 / 449,617, filed March 3, 2023; U.S. Provisional Patent Application No. 63 / 452,077, filed March 14, 2023; This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 467,045, filed May 17, 2020, U.S. Provisional Patent Application No. 63 / 429,226, filed December 1, 2022, U.S. Provisional Patent Application No. 63 / 449,610, filed March 3, 2023, and U.S. Provisional Patent Application No. 63 / 467,116, filed May 17, 2023, the entire contents of each of which are expressly incorporated by reference in their entirety.

[0002] The present disclosure relates to the fields of gene therapy and nucleic acid therapy, including compositions and methods for generating lipid nanoparticles (LNPs) that encapsulate therapeutic cargo, for example, to generate genetically modified immune effector cells. [Background technology]

[0003] Recent advances in immunotherapy combined with cell and gene therapy have demonstrated remarkable efficacy in the treatment of cancer. The development of chimeric antigen receptor (CAR) T cells is a prominent example of such a therapeutic frontier. Chimeric antigen receptors (CARs) are molecules that combine antibody-based specificity for disease-associated surface antigens with a T cell receptor activation intracellular domain that possesses disease-directed cellular immune activity. This configuration enables T cells engineered to express CARs to achieve MHC-independent primary activation via a single-chain Fv (scFv) antigen-specific extracellular domain fused to an intracellular domain that provides T cell activation and costimulatory signals. Second- and third-generation CARs also provide appropriate costimulatory signals via CD28 and / or CD137 (4-1BB) intracellular activation motifs, which enhance cytokine secretion and antitumor activity in various solid tumor and leukemia models (Pinthus, et al., 2004, J Clin Invest 114(12):1774-1781; Milone, et al., 2009, Mol Ther 17(8):1453-1464; Sadelain, et al., 2009, Curr Opin Immunol 21(2):215-223). The advantage of circumventing the need for antigen presentation by MHC molecules to achieve cytotoxicity makes CAR T cells an attractive therapeutic approach.

[0004] Adoptive cell transfer (ACT) therapy using CAR-transduced T cells has shown promise in hematological cancer trials. Currently available CAR T-cell therapies include brexucabtagene outrousel (TECARTUS®), siltacabtagene outrousel (CARVYKTI®), axicabtagene ciloleucel (YESCARTA®), and tisagenlecleucel (KYMRIAH®), which have been approved by the U.S. Food and Drug Administration to treat acute lymphoblastic leukemia (ALL), multiple myeloma, large B-cell non-Hodgkin's lymphoma, and advanced acute lymphoblastic leukemia, respectively. Other CAR T-cell therapies are being developed for other hematological cancers, including chronic lymphocytic leukemia, other forms of lymphoma, and multiple myeloma.

[0005] Therapeutic CAR T cells are prepared by first isolating natural T cells from a patient suffering from the cancer type that the CAR T cells are designed to target. The collected T cells are then typically infected with a virus encoding the CAR to target the patient's cancer type. Upon infection, the T cells display not only the appropriate antigen receptor but also the costimulatory molecules necessary to activate the T cells against the targeted antigen. These T cells are clonally expanded and then reinfused into the patient after pre-treatment chemotherapy.

[0006] Despite its remarkable efficacy in immunotherapy for cancer, CAR T-cell therapy has significant and life-threatening adverse reactions. The most common severe reaction to CAR-T therapy is cytokine release syndrome (CRS), which occurs after hundreds of millions of infused T cells release cytokines in a positive feedback loop, causing a systemic inflammatory response syndrome accompanied by fever, tachycardia, hypotension, and multi-organ dysfunction. More than 75% of patients treated with CAR-T therapy develop CRS, with a high tumor burden being the greatest risk factor. As new technologies, there is an urgent need in the art for improvements to existing cell therapies, such as CAR-based therapies, that would allow for more effective, safe, and efficient transfer of therapeutic cargo into target cells, such as T cells, B cells, natural killer (NK) cells, or dendritic cells, in vivo, in vitro, or ex vivo. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Pinthus, et al., 2004, J Clin Invest 114(12):1774-1781 [Non-patent document 2] Milone et al., 2009, Mol Ther 17(8):1453- 1464 [Non-patent document 3] Sadelain et al., 2009, Curr Opin Immunol 21(2):215-223 Summary of the Invention [Means for solving the problem]

[0008] The present disclosure generally relates to a method for producing genetically modified cells that have extended blood circulation time (e.g., increased blood T) with increased targeting ability to specific cell types (e.g., immune effector cells such as T cells, B cells, NK cells, and dendritic cells), useful for generating genetically modified cells in vivo and / or ex vivo. 1 / 2 The present invention relates to LNP compositions that exhibit physiological characteristics of increased cellular activity (e.g., increased cellular activity). LNPs can encapsulate various types of cargo, such as nucleic acids encoding desired therapeutic proteins (e.g., chimeric antigen receptors, enzymes, antibodies, etc.) or nucleic acids with sequences for gene / base editing templates. Nucleic acid molecules can be various forms of double-stranded DNA, single-stranded DNA, or RNA (mRNA, siRNA).

[0009] According to one aspect, the present disclosure provides a stealth lipid nanoparticle (LNP) comprising: (a) a therapeutic nucleic acid (TNA); (b) an ionizable lipid; (c) a sterol; (d) a first lipid-anchored polymer; and (e) a second lipid-anchored polymer, optionally wherein the second lipid-anchored polymer comprises a reactive moiety, and wherein the first lipid-anchored polymer and the second lipid-anchored polymer each comprise a lipid linker and a hydrophilic polymer.

[0010] According to some embodiments, the reactive moiety of the second lipid-anchored polymer is located on the exterior of the LNP. According to further embodiments of the aspects and embodiments herein, the stealth LNP further comprises a linker between the second lipid-anchored polymer and the reactive moiety. According to other further embodiments of the aspects and embodiments herein, the stealth LNP further comprises a covalent linker between the second lipid-anchored polymer and the reactive moiety. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is maleimide or thiol. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is maleimide. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is thiol. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is a click chemistry reagent. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is azide or DBCO. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is azide. According to some embodiments of any of the above aspects and embodiments, the reactive moiety is DBCO.

[0011] According to another aspect, the present disclosure provides a stealth lipid nanoparticle (LNP) comprising: (a) a therapeutic nucleic acid (TNA); (b) an ionizable lipid; (c) a sterol; (d) a first lipid-anchored polymer; and (e) a second lipid-anchored polymer, wherein the second lipid-anchored polymer is conjugated to a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer each comprise a lipid linker and a hydrophilic polymer. According to some embodiments, the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is selected from the group consisting of a protein, a nucleic acid, and a sugar. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an antibody, an antibody fragment, or an antibody derivative. According to some embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single-domain antibody, a single-chain antibody, and a VHH. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is an scFv. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is a VHH. According to further embodiments, the VHH is a nanobody. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is located on the exterior of the LNP. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an aptamer. According to some embodiments of any of the above aspects and embodiments, the targeting moiety specifically binds to a T cell antigen. According to some embodiments of any of the above aspects and embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR. According to some embodiments of any of the above aspects and embodiments, the targeting moiety isThe stealth LNP binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7. According to some embodiments of any of the above aspects and embodiments, the stealth LNP further comprises a linker between the second lipid-anchored polymer and the targeting moiety. According to some embodiments of any of the above aspects and embodiments, the first lipid linker and the second lipid linker are each independently selected from the group consisting of a non-ester-containing linker and an ester-containing linker. According to some embodiments of any of the above aspects and embodiments, the ester-containing linker is selected from the group consisting of an amide linker and a carbamate linker. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is conjugated to the second lipid-anchored polymer via maleimide conjugation. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is conjugated to the second lipid-anchored polymer via click chemistry. According to some embodiments of any of the above aspects and embodiments, the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, derivatives thereof, and combinations thereof. According to some embodiments of any of the above aspects and embodiments, the sterol is cholesterol. According to some embodiments of any of the above aspects and embodiments, the sterol is beta-sitosterol. According to some embodiments of any of the above aspects and embodiments, the ionizable lipid is selected from the group consisting of 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), DLin-MC3-DMA,N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), Cero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamidoethyl-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint Lipids (e.g., SAINT-2, N-methyl 1,2-Dioleoyl-4-(dioleyl)methylpyridinium), 1,2-Dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-Dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,2-Dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), Dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), Dioleyldimethylammonium chloride (DODAC), 1-Palmitoyl- In some variations, the condensing agent, e.g., the cationic lipid, is selected from the group consisting of 2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC) and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g., the cationic lipid, is selected from the group consisting of dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA),Lipids such as heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen), SM102, L369, LP01, "SS-cleavable lipids," and mixtures thereof. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a lipid comprising at least one hydrophobic tail. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a lipid comprising at least two hydrophobic tails. According to some embodiments of any of the above aspects and embodiments, each hydrophobic tail comprises at least 18 carbon atoms (C, 18 According to some embodiments of any of the above aspects and embodiments, each hydrophobic tail comprises a carbon chain having 18 to 22 carbon atoms (C 18 -C 22 According to some embodiments of any of the above aspects and embodiments, each hydrophobic tail comprises a carbon chain having 18 carbon atoms (C 18According to some embodiments of any of the above aspects and embodiments, the first lipid linker and the second lipid linker each independently comprise 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphine dioleoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), and combinations and derivatives thereof. According to some embodiments of any of the above aspects and embodiments, the first lipid linker and the second lipid linker are each independently selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and combinations thereof. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer are each independently DSPE, DODA, DSG, or combinations thereof.According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a polymer selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polyoxazoline (POZ), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyamide, and combinations thereof. According to some embodiments, the polymer is PEG. According to some embodiments of any of the above aspects and embodiments, the PEG is selected from the group consisting of PEG2000, PEG2000Ome, and PEG2000-OH. According to some embodiments, the polymer is polyglycerol (PG). According to some embodiments of any of the above aspects and embodiments, the PG comprises at least 5-60 glycerol units, e.g., at least 5-50, 10-50, 20-50, 25-50, 40-50, 10-20, 10-30, 10-40, 20-40, 20-30, 30-40, or 40-50 glycerol units. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer. The first and second lipid anchor polymers each independently comprise DSPE, DODA, DSG, or a combination thereof. According to some embodiments of any of the above aspects and embodiments, the first and second lipid anchor polymers each independently comprise DSPE-PEG, DODA-PG, DSPE-PG, DODA-PEG, DSG-PEG, DSG-PG, or a combination thereof. According to some embodiments of any of the above aspects and embodiments, the first and second lipid anchor polymers each comprise different lipid linkers. According to some embodiments of any of the above aspects and embodiments, the first and second lipid anchor polymers each comprise the same lipid linker. According to some embodiments of any of the above aspects and embodiments, the first and second lipid anchor polymers are different. According to some embodiments of any of the above aspects and embodiments, the first and second lipid anchor polymers are the same. According to some embodiments of any of the above aspects and embodiments, both the first and second lipid anchor polymers are DSPE-PEG. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer and the second lipid-anchored polymer are both DODA-PG.

[0012] According to some embodiments of any of the above aspects and embodiments, the stealth LNP further comprises a helper lipid. According to some embodiments of any of the above aspects and embodiments, the helper lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), dioleoyl-sn-glycero-phospho ... di-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin sucralose (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,The helper lipid is selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, DODA, ceramide, and derivatives and combinations thereof. According to some embodiments of any of the above aspects and embodiments, the helper lipid is DSPC.

[0013] According to some embodiments of any of the above aspects and embodiments, the ionizable lipid is present in a molar percentage of about 30% to about 80%, e.g., about 30% to about 70%, about 40% to about 80%, about 50% to about 80%, about 50% to about 60%, about 30% to about 50%, or about 40% to about 60%. According to some embodiments of any of the above aspects and embodiments, the sterol is present in a molar percentage of about 20% to about 50%, e.g., about 20% to about 45%, about 25% to about 50%, about 30% to about 45%, about 35% to about 50%, about 40% to about 45%, or about 45% to about 50%. According to some embodiments of any of the above aspects and embodiments, the sterol is present in a molar percentage of about 35% to about 40%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer and the second anchor polymer are present in a combined molar percentage of about 1% to about 8%, e.g., 1, 2, 3, 4, 5, 6, 7, or 8%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer and the second anchor polymer are present in a combined molar percentage of about 2% to about 5%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer and the second anchor polymer are present in a combined molar percentage of about 3%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer is present in a molar percentage of about 1% to about 7%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer is present in a molar percentage of about 1.5% to about 5%. According to some embodiments of any of the above aspects and embodiments, the first lipid anchor polymer is present in a molar percentage of about 2% to about 3%. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer is present in a molar percentage of about 2% to about 3%. According to some embodiments of any of the above aspects and embodiments, the first lipid-anchored polymer is present in a molar percentage of about 2.5%.According to some embodiments of any of the above aspects and embodiments, the second lipid-anchored polymer is present at a molar percentage of about 0.25% to about 1%. According to some embodiments of any of the above aspects and embodiments, the second lipid-anchored polymer is present at a molar percentage of about 0.35% to about 0.75%. According to some embodiments of any of the above aspects and embodiments, the second lipid-anchored polymer is present at a molar percentage of about 0.5%. According to some embodiments of any of the above aspects and embodiments, the helper lipid is present at a molar percentage of about 2% to about 20%, e.g., about 2% to about 5%, about 10% to about 20%, about 5% to about 10%, about 2% to about 10%, about 5% to about 20%, or about 15% to about 20%. According to some embodiments of any of the above aspects and embodiments, the helper lipid is present at a molar percentage of about 10%.

[0014] According to some embodiments of any of the above aspects and embodiments, the stealth LNP further comprises an immunosuppressant.

[0015] According to some embodiments of any of the above aspects and embodiments, the nanoparticles have a ratio of total lipids to TNA of about 10:1 to about 40:1, e.g., about 10:1 to about 40:1, about 15:1 to about 40:1, about 20:1 to about 40:1, about 25:1 to about 40:1, about 30:1 to about 40:1, about 35:1 to about 40:1, about 20:1 to about 30:1, about 15:1 to about 35:1, about 15:1 to about 30:1, or about 20:1 to about 25:1. According to some embodiments of any of the above aspects and embodiments, the LNPs have a diameter of about 40 nm to about 120 nm. According to some embodiments of any of the above aspects and embodiments, the LNPs have a diameter of less than about 100 nm, e.g., less than about 90 nm, less than about 80 nm, less than about 70 nm, less than about 60 nm, less than about 50 nm, less than about 40 nm, less than about 30 nm, or less than about 20 nm. According to some embodiments of any of the above aspects and embodiments, the LNPs have a diameter of about 60 nm to about 80 nm. According to some embodiments of any of the above aspects and embodiments, the LNPs are present in an LNP composition comprising a plurality of LNPs having an average diameter of about 40 nm to about 120 nm. According to some embodiments of any of the above aspects and embodiments, the LNPs are present in an LNP composition comprising a plurality of LNPs having an average diameter of less than about 100 nm. According to some embodiments of any of the above aspects and embodiments, the LNPs are present in an LNP composition comprising a plurality of LNPs having an average diameter of about 60 nm to about 80 nm. According to some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of RNA, DNA, and derivatives and analogs thereof. According to some embodiments of any of the above aspects and embodiments, the TNA encodes a therapeutic gene and / or a therapeutic protein. According to some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of mRNA, siRNA, synthetic ribozyme, antisense RNA, and gRNA. According to some embodiments of any of the above aspects and embodiments, the TNA is mRNA.According to some embodiments of any of the above aspects and embodiments, the TNA is selected from the group consisting of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). According to some embodiments of any of the above aspects and embodiments, the TNA is ssDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is linear ssDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is dsDNA. According to some embodiments of any of the above aspects and embodiments, the TNA is a capsid-free non-viral DNA vector (ceDNA vector) with covalently closed ends. According to some embodiments of any of the above aspects and embodiments, the TNA encodes a chimeric antigen receptor (CAR). According to some embodiments of any of the above aspects and embodiments, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain. According to some embodiments of any of the above aspects and embodiments, the signaling domain is a CD3 zeta signaling domain. According to some embodiments of any of the above aspects and embodiments, the antigen-binding domain is an antibody or an antigen-binding fragment thereof. According to some embodiments, the antigen-binding fragment is a Fab, Fab', scFv, or VHH. According to some embodiments of any of the above aspects and embodiments, the antigen-binding domain binds to a tumor antigen. According to some embodiments, the tumor antigen is associated with a hematological malignancy. According to some embodiments, the tumor antigen is associated with a solid tumor. According to some embodiments of any of the above aspects and embodiments, the costimulatory signaling region comprises an intracellular domain of a costimulatory molecule selected from the group consisting of CD13, CD19, CD21, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.According to some embodiments of any of the above aspects and embodiments, the TNA is synthetically produced in a cell-free environment. According to some embodiments of any of the above aspects and embodiments, the TNA encodes a therapeutic gene and / or a therapeutic protein.

[0016] According to another aspect, the present disclosure provides a cell comprising the stealth LNP of any one of the aspects and embodiments herein. According to some embodiments, the cell is in vitro, ex vivo, or in vivo. According to some embodiments, the cell is in vitro. According to some embodiments, the cell is ex vivo. According to some embodiments, the cell is in vivo. According to some embodiments, the cell is a T cell. According to some embodiments of any of the above aspects and embodiments, the cell is an autologous T cell. According to some embodiments, the cell is an allogeneic T cell.

[0017] According to another aspect, the present disclosure provides a pharmaceutical composition comprising the stealth LNP of any one of the aspects and embodiments herein or the cell of any one of the aspects and embodiments herein. According to some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or carrier. According to some embodiments, the pharmaceutical composition further comprises an immunosuppressant.

[0018] According to some embodiments of any of the above aspects and embodiments, the pharmaceutical composition further comprises a tyrosine kinase inhibitor (TKI). According to some embodiments, the tyrosine kinase inhibitor is a pharmaceutically acceptable salt of the TKI.

[0019] According to some aspects, the present disclosure provides a method of treating a disease or disorder in a subject, the method comprising administering to the subject a therapeutically effective amount of a stealth LNP of any one of the aspects and embodiments herein, a cell of any one of the aspects and embodiments herein, and / or a pharmaceutical composition of any one of the aspects and embodiments herein.

[0020] According to some embodiments of any of the above aspects and embodiments, the disease or disorder is a genetic disease or disorder. According to some embodiments of any of the above aspects and embodiments, the genetic disease or disorder is sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS IS), Hurler-Scheie syndrome (MPS I HS), Hunter syndrome (MPS II), Sanfilippo types A, B, C, and D (MPS III), A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPS IX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I, II, and III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency) ), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency,The disease or disorder is selected from the group consisting of generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is hemophilia A. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is hemophilia B. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is phenylketonuria (PKU). According to some embodiments of any of the above aspects and embodiments, the disease or disorder is Wilson's disease. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is Gaucher disease types I, II, and III. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is Stargardt macular dystrophy. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is LCA10. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is Usher syndrome. According to some embodiments of any of the above aspects and embodiments, the disease or disorder is wet AMD.

[0021] According to some aspects, the present disclosure provides a method of delivering a therapeutic nucleic acid (TNA) to a subject, the method comprising administering to the subject a therapeutically effective amount of a stealth LNP of any one of the aspects and embodiments herein, a cell of any one of the aspects and embodiments herein, and / or a pharmaceutical composition of any one of the aspects and embodiments herein.

[0022] According to some aspects, the present disclosure provides a method of delivering a therapeutic gene and / or a therapeutic protein, wherein the therapeutic gene and / or protein is encoded by a therapeutic nucleic acid (TNA), to a cell, comprising contacting the cell in a subject with a stealth LNP of any one of the aspects and embodiments herein and / or a pharmaceutical composition of any one of the aspects and embodiments herein, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

[0023] According to some aspects, the present disclosure provides a method of delivering a therapeutic gene to the nucleus of a cell, comprising contacting the cell with a stealth LNP of any one of the aspects and embodiments herein and / or the pharmaceutical composition of any one of the aspects and embodiments herein, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell.

[0024] According to some embodiments of any of the above aspects and embodiments, the cells are in vitro. According to some embodiments of any of the above aspects and embodiments, the cells are in vivo. According to some embodiments of any of the above aspects and embodiments, the cells are ex vivo.

[0025] According to another aspect, the present disclosure provides a method of providing anti-tumor immunity to a subject, the method comprising administering to the subject a stealth LNP of any one of the aspects and embodiments herein, a cell of any one of the aspects and embodiments herein, and / or a pharmaceutical composition of any one of the aspects and embodiments herein, thereby providing anti-tumor immunity to the subject.

[0026] According to another further aspect, the present disclosure provides a method of treating a subject having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a stealth LNP of any one of the aspects and embodiments herein, a cell of any one of the aspects and embodiments herein, and / or a pharmaceutical composition of any one of the aspects and embodiments herein, thereby treating the subject.

[0027] According to some embodiments of any of the above aspects and embodiments, the cell is a T cell. According to some embodiments of any of the above aspects and embodiments, the cell is an autologous T cell. According to some embodiments of any of the above aspects and embodiments, the cell is an allogeneic T cell. According to some embodiments of any of the above aspects and embodiments, the subject is a human.

[0028] According to some aspects, the present disclosure provides a method for producing a stealth LNP comprising a targeting moiety, the method comprising: (a) providing a stealth LNP according to any one of the aspects and embodiments herein, wherein the second lipid-anchored polymer comprises a first reactive moiety; (b) providing a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety are capable of reacting to form a covalent bond; and (c) contacting the stealth LNP of (a) with the targeting moiety of (b) under conditions sufficient to allow reaction between the first reactive moiety and the second reactive moiety, thereby producing a stealth LNP comprising the targeting moiety. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is a maleimide and the second reactive moiety is a thiol. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is a thiol and the second reactive moiety is a maleimide. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety and the second reactive moiety are click chemistry reagents. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is azide and the second reactive moiety is DBCO. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is DBCO and the second reactive moiety is azide. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is selected from the group consisting of a protein, a nucleic acid, and a sugar. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an antibody, an antibody fragment, or an antibody derivative. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single-domain antibody, a single-chain antibody, and a VHH.According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is an scFv. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is a VHH. According to further embodiments, the VHH is a nanobody. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an aptamer. According to some embodiments of any of the above aspects and embodiments, the targeting moiety specifically binds to a T cell antigen. According to some embodiments of any of the above aspects and embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR. According to some embodiments of any of the above aspects and embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.

[0029] According to some aspects, the present disclosure provides a kit for preparing a targeted stealth LNP, the kit including: (a) the stealth LNP of any one of the aspects and embodiments herein, wherein the second lipid-anchored polymer comprises a first reactive moiety; and (b) instructions for producing the targeted stealth LNP by contacting the stealth LNP of (a) with a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety are capable of reacting to form a covalent bond.

[0030] According to another aspect, the present disclosure provides a kit for producing targeted stealth LNPs, the kit including: (a) a stealth LNP of any one of the aspects and embodiments herein, wherein the second lipid-anchored polymer comprises a first reactive moiety; (b) a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety are capable of reacting with each other to form a covalent bond; and (c) instructions for producing the targeted stealth LNPs by contacting the stealth LNP of (a) with the targeting moiety of (b).

[0031] According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is a maleimide and the second reactive moiety is a thiol. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is a thiol and the second reactive moiety is a maleimide. According to some embodiments of any of the above aspects and embodiments, the second reactive moiety is a click chemistry reagent. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is an azide and the second reactive moiety is DBCO. According to some embodiments of any of the above aspects and embodiments, the first reactive moiety is DBCO and the second reactive moiety is azide. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is selected from the group consisting of a protein, a nucleic acid, and a sugar. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an antibody, an antibody fragment, or an antibody derivative. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single-domain antibody, a single-chain antibody, and a VHH. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is an scFv. According to some embodiments of any of the above aspects and embodiments, the antibody, antibody fragment, or antibody derivative is a VHH. According to further embodiments, the VHH is a nanobody. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative. According to some embodiments of any of the above aspects and embodiments, the targeting moiety is an aptamer. According to some embodiments of any of the above aspects and embodiments, the targeting moiety specifically binds to a T cell antigen.According to some embodiments of any of the above aspects and embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR. According to some embodiments of any of the above aspects and embodiments, the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.

[0032] According to another aspect, the present disclosure provides a stealth lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), an ionizable lipid number 87, cholesterol, a first lipid-anchored polymer, and a second lipid-anchored polymer, wherein the first lipid-anchored polymer is DSG-PEG2000-OMe and the second lipid-anchored polymer is DSPE-PEG5000-maleimide reactive moiety. According to some embodiments, the stealth LNP comprises the therapeutic nucleic acid (TNA), about 57.5 mol% of the ionizable lipid number 87, about 39.5 mol% of cholesterol, about 2.5 mol% of the first lipid-anchored polymer, and about 0.5 mol% of the second lipid-anchored polymer.

[0033] According to some aspects, the present disclosure provides a stealth lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), an ionizable lipid number 87, DSPC, cholesterol, a first lipid anchor polymer, and a second lipid anchor polymer, wherein the first lipid anchor polymer is DSG-PEG2000-OMe and the second lipid anchor polymer is DSPE-PEG5000-maleimide reactive moiety. According to some embodiments, the stealth LNP comprises the therapeutic nucleic acid (TNA), about 47.5 mol% of the ionizable lipid number 87, about 10% of DSPC, about 39.5 mol% of cholesterol, about 2.5 mol% of the first lipid anchor polymer, and about 0.5 mol% of the second lipid anchor polymer. According to some embodiments, the stealth LNP comprises a therapeutic nucleic acid (TNA), about 47.5 mol % ionizable lipid, about 10 mol % helper lipid, about 39.5 mol % sterol, about 2.5 mol % first lipid-anchored polymer, and about 0.5 mol % second lipid-anchored polymer comprising a reactive moiety for attaching a targeting moiety. [Brief explanation of the drawings]

[0034] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to illustrative embodiments of the disclosure that are depicted in the accompanying drawings. However, because the present disclosure may admit of other equally effective embodiments, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope.

[0035] [Figure 1]Illustrated are three different stealth LNPs of the present disclosure. The first is a stealth LNP that displays reactive species on the surface but has not yet reacted with a targeting moiety. The middle LNP is stealth LNP1, which contains 57.5 mol% ionizable lipid number 87, 39.5 mol% cholesterol as the structural lipid, 2.5 mol% lipid-anchored polymer 1 (i.e., DSG-PEG2000-OMe), and 0.5% lipid-anchored polymer 2 (i.e., DSPE-PEG5000-maleimide reactive species) conjugated to an scFv. The third stealth LNP on the right is composed of 47.5 mol% ionizable lipid number 87, 10 mol% helper lipid (i.e., DSPC), 39.5 mol% cholesterol structural lipid, 2.5 mol% lipid anchor 1 (i.e., DSP-PEG2000-OMe), and 0.5 mol% lipid anchor polymer 2 as DSPE-PEG5K-maleimide reacted with an scFv targeting moiety. [Figure 2] 1 illustrates a stealth LNP with an ScFv conjugated to the surface of the LNP, where the conjugated ScFv protrudes into the biological environment and can target the LNP to a specific surface antigen on a predetermined cellular target. [Figure 3] Unconjugated Stealth LNP is 0.3mpk Trilink m1

number

number

[0023] Figure 10 is a panel of graphs showing that stealth LNPs bearing anti-CD3 scFv, anti-CD5 scFv, anti-CD6 scFv, anti-CD7 scFv, and trastuzumab (anti-Her) all exhibited dose-dependent binding, uptake, and mRNA cargo expression. Anti-HER2 antibody was included as a negative control because HER2 was not found in these cells, confirming that even non-targeted LNPs remained stealthy. [Figure 15] 1 is a panel of flow cytometry results showing that stealth-targeted LNPs with anti-CD3-scFv, anti-CD5-scFv, anti-CD6 scFv, and anti-CD7 scFv exhibited clear binding and were internalized by resting primary human T cells, but exhibited reduced cargo expression. [Figure 16] FIG. 10 is a graph showing that much less mRNA was detected in resting T cells compared to anti-CD3 LNPs that autoactivated T cells when bound to and entered the cells. [Figure 17] Figure 12 is a graph showing that anti-CD3 LNPs activated T cells when bound to the cells during overnight incubation with primary human T cells in vitro. CD69 is a marker of early T cell activation. 40-fold greater T cell activation was observed in the presence of anti-CD3 LNPs compared to T cell activation with anti-CD5 LNPs, anti-CD6 LNPs, or anti-CD7 LNPs. [Figure 18]Conjugates were prepared using the maleimide conjugation protocol described in the Examples, and the conjugated LNPs were incubated with resting and activated T cells. The graph shows % DiD uptake on the Y-axis and green lantern mRNA expression on the X-axis. The data were highly repeatable across two donors. [Figure 19] Results are shown from experiments comparing T cell-targeted LNPs for their ability to target, bind to, enter, and express their mRNA cargo to human T cells in humanized mice. [Figure 20] 1 shows the results of an in vivo study in which LNP2 conjugated to anti-CD7 scFv exhibited highly selective receptor-mediated uptake and mRNA expression in humanized mice upon systemic administration. [Figure 21] 1 shows dose-dependent receptor-mediated delivery and expression of mRNA in vivo using humanized mice. [Figure 22] 1 shows a graph of quantification via qPCR of ceDNA copies in whole blood of CD-1 mice treated with LNP201, LNP202, and LNP203 at 0, 1, 3, 6, and 24 hours post-administration. [Figure 23A] The HPLC-SEC readouts of LNP formulations with incremental mol% of the first lipid-anchored polymer (i.e., LNPs with 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 5 mol%, and 7 mol% DSG-PEG2000-OMe) show different retention times. [Figure 23B] Retention times for LNP formulations with 1.5 mol% lipid-anchored polymer (DSG-PEG2000-OMe) are shown (wavelength readings: 214 nm to track lipids and 260 nm to track nucleic acid cargo). [Figure 23C] Retention times for LNPs with 7 mol% lipid-anchored polymer (DSG-PEG2000-OMe) are shown (wavelength readings: 214 nm to track lipids and 260 nm to track nucleic acid cargo). [Figure 24]Various conjugation chemistry schemes are shown. [Figure 25] 1 shows a workflow for screening and comparing various LNP formulations for their ability to enter cells without endocytosis inhibitors using primary human hepatocytes. [Figure 26A] Figure 25 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH: "A05") conjugation to target hepatic ASGPR1 protein for their relative ability to enter primary human hepatocytes after 24 hours, following the workflow shown in Figure 25. [Figure 26B] Same as above [Figure 27A] Figure 25 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH: "A05") conjugation to target hepatic ASGPR1 protein for their relative ability to express mLuc and rLuc cargo, following the workflow shown in Figure 25. [Figure 27B] Same as above [Figure 28] 1 shows a workflow for screening and comparing various LNP formulations for their relative ability to enter cells in the presence of an endocytosis inhibitor (DynGo-4a) using primary human hepatocytes. [Figure 29A] Figure 28 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH ("A05") and scFv) conjugation to target hepatic ASGPR1 protein for their relative ability to enter primary human hepatocytes after 24 hours, following the workflow shown in Figure 28. [Figure 29B] Same as above [Figure 30A] Figure 28 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH ("A05") and scFv) conjugation to target hepatic ASGPR1 protein for their relative ability to express mLuc cargo under various inhibitory conditions, following the workflow shown in Figure 28. [Figure 30B] Same as above [Figure 31A]Figure 28 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH: "A05") conjugation to target hepatic ASGPR1 protein for their relative ability to enter primary human hepatocytes after 24 hours, following the workflow shown in Figure 28. [Figure 31B] Same as above [Figure 32A] Figure 28 shows the results of a screening study of LNP formulations of the present disclosure having antibody (VHH) conjugation to target hepatic ASGPR1 protein for their relative ability to express mLuc and rLuc cargo, following the workflow shown in Figure 28. [Figure 32B] Same as above DETAILED DESCRIPTION OF THE INVENTION

[0036] The present disclosure provides lipid nanoparticles (LNPs) and LNP compositions (e.g., pharmaceutical compositions) comprising a therapeutic nucleic acid (TNA), e.g., a gene expression vector such as closed-end DNA (ceDNA), a single-stranded DNA vector, or a messenger RNA (mRNA), where the structural LNP component comprises an ionizable lipid, with or without a "helper" lipid, a structural lipid, e.g., a sterol, and one or more types of lipid anchor polymers comprising a hydrophilic polymer (e.g., PEG or polyglycerol), a lipid moiety having at least one hydrophobic tail having 16 to 22 carbon atoms in a single aliphatic chain backbone, and a linker connecting the polymer to the lipid moiety.

[0037] The LNPs disclosed herein offer surprising and unexpected "stealth" properties compared to previously known LNPs. For example, helper lipids, when present, function to increase the membrane fusogenicity of the LNP's lipid bilayer and facilitate endosomal escape; structural lipids in the LNP contribute to the membrane integrity and stability of the LNP; and lipid-anchored polymers in the LNP can inhibit LNP aggregation and provide steric stabilization (e.g., enhancing the stealth properties of the overall LNP characteristics in the circulation (e.g., blood compartment) by minimizing interactions between opsonins present in the blood and the surface of the LNP). In addition, the present disclosure provides lipid-anchored polymers in which the number of aliphatic carbons in the lipid moiety of the lipid-anchored polymer is important for slowing dissociation of the lipid-anchored polymer away from the LNP, allowing the LNP to remain intact and avoid nonspecific fusion or clearance within the first hour in the blood or plasma compartment. The present disclosure provides LNPs in which at least one of the lipids in the lipid-anchored polymer contains 16, 18, or 20 aliphatic carbons to more firmly anchor the lipid-anchored polymer to the LNP. In some embodiments, at least one lipid in the lipid-anchored polymer with at least 18 aliphatic carbons is useful for creating stealth LNPs. In other embodiments, at least one lipid in the lipid-anchored polymer with at least 20 aliphatic carbons is useful for creating stealth LNPs.

[0038] The present disclosure also provides "cell-targeted stealth LNPs" by combining the above-described stealth properties with cell targeting of the LNPs through conjugation of a targeting moiety to one of the lipid-anchored polymers in the LNP. Furthermore, the disclosed stealth cell-targeting LNP compositions can further include a targeting moiety, such as a single-chain fragment variable antibody (scFv) and / or a single-domain antibody (VHH), linked to the LNP, where the scFv or VHH is directed against an antigen present on the surface of a cell (e.g., tumor cell, T cell, B cell, NK cell, etc.), thereby improving the targeting specificity of the stealth LNP to a desired tissue or cell type. The stealth-targeting LNP compositions described herein advantageously provide efficient covalent conjugation with little or no effect on blood pharmacokinetics (PK), particle size, and stability compared to unconjugated stealth LNPs. It is a finding of the present disclosure that DBCO-mediated conjugation (via "click chemistry") or maleimide conjugation (via riol-maleimide reaction) between a targeting moiety (e.g., an scFv or VHH) and a lipid-anchored polymer present on the surface of stealth LNPs results in a robust linkage that maintains the physiochemical properties of stealth LNPs, and that the resulting stealth LNPs containing targeting moieties effectively demonstrated significantly improved specificity and targeting efficiency to desired cell types in vivo.

[0039] The present disclosure also provides stealth LNP compositions comprising lipid-anchored polymers bearing reactive species, e.g., maleimides, azides, etc., that can react with targeting moieties functionalized with thiol (-SH) reactive species or dibenzocyclooctyne (DBCO) reactive species.

[0040] I. Definition As used herein, the term "activated" refers to a state of T cells that have been stimulated sufficiently to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0041] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a set of polypeptides, typically two in the simplest embodiment, that, in the case of an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and provides intracellular signal generation. In some embodiments, the CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an intracellular signaling domain) that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some aspects, the set of polypeptides are contiguous with one another. In some embodiments, the set of polypeptides comprises a dimerization switch that, in the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., link the antigen-binding domain to the intracellular signaling domain. In some aspects, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In some aspects, the costimulatory molecule is selected from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, where the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0042] A CAR comprising an antigen binding domain (e.g., scFv, VHH, or TCR) that targets a specific tumor marker X, such as those described herein, is also referred to as an XCAR.

[0043] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting intracellular information and regulating cellular activity through defined signaling pathways, either by generating second messengers or by functioning as an effector by responding to such messengers.

[0044] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers and fragments of immunoglobulin molecules. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFvs), Fd fragments consisting of a VH domain and a CHI domain, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0045] As used herein, the term "antigen" refers to any foreign substance that induces an immune response in the body.

[0046] The term "camelized" VH refers to an ISVD in which one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody are replaced by one or more of the amino acid residues that occur at the corresponding positions in the VHH domain of a heavy-chain antibody. Such "camelized" substitutions may be inserted at amino acid positions forming the VH-VL interface and / or at amino acid positions present at the VH-VL interface and / or at so-called camelid-characteristic residues, as defined herein (see also, e.g., WO9404678 and Davies and Riechmann (1994 and 1996)). Reference is made to Davies and Riechmann (FEBS 339:285-290, 1994; Biotechnol. 13:475-479, 1995; Prot. Eng. 9:531-537, 1996) and Riechmann and Muyldermans (J. Immunol. Methods 231:25-38, 1999).

[0047] The terms "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, regardless of the number of transformations. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutation. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a clear designation is intended, this will be clear from the context.

[0048] The term "CDR region" refers to an antibody complementarity determining region (CDR) defined by any one of the methods commonly used to define antibody CDRs, and may further include up to one amino acid at the N-terminus of the defined CDR or up to three amino acids at the C-terminus of the defined CDR.

[0049] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0050] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, ceDNA, ssDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of the gene or cDNA can be considered to encode the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Protein- and RNA-encoding nucleotide sequences can contain introns.

[0051] As used herein, the term "epitope" is defined in the context of the molecular interactions between an antibody (e.g., IgG, scFv, VHH, etc.) and its corresponding "antigen" (Ag). Generally, "epitope" refers to the area or region on an Ag that is specifically recognized and bound by an antibody (e.g., IgG, scFv, VHH, etc.), i.e., the area or region that is in physical contact with the antibody (e.g., IgG, scFv, VHH, etc.). Physical contact is achieved by the formation of a human-like V H The distance criteria for atoms in H and Ag molecules (e.g., a distance cutoff of 4 Å) can be defined. The physical contact and distance criteria between an antibody or other binding molecule and a target antigen can be determined by protein crystallography of the antibody-antigen complex.

[0052] As used herein, the term "Fc domain" refers to the C domain of an antibody. H 2 domain and C H A crystallizable fragment domain or region obtained from an antibody containing three domains. In antibodies, two Fc domains are connected by two or more disulfide bonds and C H The three domains are held together by hydrophobic interactions. The Fc domain can be obtained by digesting an antibody with the protease Pain.

[0053] The term "immunoglobulin single-chain variable domain" (abbreviated herein as "ISVD" and used interchangeably with "single variable domain") defines a molecule in which the antigen-binding site resides on and is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins or fragments thereof, in which two immunoglobulin domains, in particular two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form the antigen-binding site. In the latter case, the complementarity-determining regions (CDR) regions of both the VH and the VL contribute to the antigen-binding site, i.e., a total of six CDRs are involved in forming the antigen-binding site. In view of the above definitions, the antigen-binding domain of a conventional four-chain antibody (e.g., an IgG, IgM, IgA, IgD, or IgE molecule known in the art), or an Fv fragment such as a Fab fragment, a F(ab)2 fragment, a disulfide-linked Fv or scFv fragment, or a diabody derived from such a conventional four-chain antibody (all known in the art) would not normally be considered an ISVD; in these cases, binding to a respective epitope of an antigen would typically occur not by one (single) immunoglobulin domain, but by a pair (associated) immunoglobulin domains, such as a light chain variable domain and a heavy chain variable domain, which together bind to the respective epitope of the antigen, i.e., a VH-VL pair of immunoglobulin domains.

[0054] In contrast, an ISVD can specifically bind to an epitope of an antigen without pairing with an additional immunoglobulin variable domain. The binding site of an ISVD is formed by a single VHH domain or VH domain. Therefore, the antigen-binding site of an ISVD is formed by three or fewer CDRs. Thus, the single variable domain may be a heavy chain variable domain sequence (e.g., a Vs sequence or a VHH sequence) or a suitable fragment thereof, as long as it can form a single antigen-binding unit (i.e., a functional antigen-binding unit essentially consisting of a single variable domain such that the single antigen-binding domain does not need to interact with another variable domain to form a functional antigen-binding unit). As used herein, ISVD refers to VHH, human-like VHH, and camelized VHH. H is selected from the group consisting of:

[0055] As used herein, the terms "NANOBODY" and "NANOBODIES" are registered trademarks of Ablynx NV. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single-chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., with respect to the N- and C-termini of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0056] One or more lipid-anchored polymers of the lipid nanoparticles (LNPs) of the present disclosure may be chemically conjugated to an scFv or VHH against an epitope.

[0057] The portion of the CAR of the present disclosure that comprises an antibody or antibody fragment thereof can exist in various forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition of the present disclosure comprises an antibody fragment. In further embodiments, the CAR comprises an antibody fragment that comprises an scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of several well-known numbering schemes, including the scheme described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), the scheme described in Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or a combination thereof.

[0058] As used herein, the term "binding domain" or "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0059] The portion of the CAR of the present disclosure that comprises an antibody or antibody fragment thereof can exist in various forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR composition of the present disclosure comprises an antibody fragment. In further embodiments, the CAR comprises an antibody fragment that comprises an scFv.

[0060] As used herein, the term "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in an antibody molecule, in their naturally occurring configuration, and usually determines the class to which the antibody belongs.

[0061] As used herein, the term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) light chain and lambda (λ) light chain refer to the two major antibody light chain isotypes.

[0062] As used herein, the term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term may also be taken to mean an antibody produced by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein, or an antibody produced by synthesis of an amino acid sequence specifying the antibody, where the DNA or amino acid sequence is obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0063] As used herein, the term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can function as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, as the term is used herein, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen." Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present disclosure includes the use of partial nucleotide sequences of two or more genes, with these nucleotide sequences arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that an antigen can be generated, synthesized, or derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or bodily fluids having other biological components.

[0064] As used herein, the term "anti-cancer effect" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" may also be manifested primarily by the ability of peptides, polynucleotides, cells, and antibodies to prevent the development of cancer. The term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, for example, a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0065] As used herein, the term "autologous" refers to any material originating from the same individual that is later reintroduced into the individual.

[0066] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical. In some embodiments, allogeneic materials derived from individuals of the same species may be genetically sufficiently different to interact antigenically.

[0067] The phrase "disease associated with expression of a tumor antigen described herein" includes, but is not limited to, a disease associated with expression of a tumor antigen described herein or a condition associated with cells expressing a tumor antigen described herein (including, for example, a proliferative disease (such as a cancer or a malignant tumor) or a precancerous condition (such as myelodysplasia, myelodysplastic syndrome, or preleukemia)), or a non-cancer-related indication associated with cells expressing a tumor antigen described herein. In some aspects, a cancer associated with expression of a tumor antigen described herein is a hematological cancer. In some aspects, a cancer associated with expression of a tumor antigen described herein is a solid cancer. Additional diseases associated with expression of a tumor antigen described herein include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of a tumor antigen described herein. Non-cancer-related indications associated with expression of a tumor antigen described herein include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells express, or at any time express, mRNA encoding the tumor antigen. In some embodiments, tumor antigen-expressing cells produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins can be present at normal or reduced levels. In some embodiments, tumor antigen-expressing cells produce detectable levels of tumor antigen proteins at one time, and then subsequently produce substantially no detectable tumor antigen proteins.

[0068] As used herein, the term "cancer" refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can metastasize locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc.

[0069] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex or through the appropriate NK receptor or signaling domain of the CAR. Stimulation can mediate altered expression of certain molecules.

[0070] The term "stimulatory molecule" refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides cytoplasmic signaling sequences that regulate immune cell activation upon stimulation for at least some aspects of immune cell signaling pathways. In some aspects, the signal is a primary signal that is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule and results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act upon stimulation may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM-containing cytoplasmic signaling sequences of particular use in the present disclosure include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCERIG), Fc gamma R11a, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In certain CARs of the present disclosure, the intracellular signaling domain in any one or more CARs of the present disclosure comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3-zeta. In certain CARs of the present disclosure, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 18, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In certain CARs of the present disclosure, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 20, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0071] The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0072] The term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell. Examples of immune effector function, e.g., in a CART cell, include helper activity, including cytolytic activity and cytokine secretion.

[0073] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0074] A primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITEM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma R11a, FcR beta (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

[0075] The terms "zeta," or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta," are defined as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "zeta stimulatory domain," or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain," are defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or a functional derivative thereof, sufficient to functionally transmit the initial signal required for T cell activation. In some embodiments, the zeta cytoplasmic domain comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., that is a functional ortholog thereof.

[0076] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl 1d, ITGAE, CD103, ITGAL, CDl 1a, LFA-1, ITGAM, CDl 1b, ITGAX, CDl 1c, and ITGB. Ligands that specifically bind to CD1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0077] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

[0078] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0079] "Immune effector cells," as that term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0080] "Immune effector function or immune effector response," as that term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes killing of a target cell or promotes inhibition of its growth or proliferation. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0081] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that a nucleotide sequence that encodes a protein may, in some versions, contain introns.

[0082] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition described herein that is effective to achieve a particular biological result.

[0083] The term "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

[0084] The term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue, or system.

[0085] The term "transfer vector" refers to a composition that contains an isolated nucleic acid (e.g., ceDNA, ssDNA, mRNA) and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, lipid nanoparticles, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, lentiviral vectors, and the like.

[0086] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including double-stranded ceDNA, ssDNA, mRNA, cosmids, plasmids (e.g., naked plasmids or plasmids contained in liposomes or LNPs), and viruses that incorporate the recombinant polynucleotide (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses (AAV)).

[0087] As used herein, the terms "homology" or "identity" refer to the subunit sequence identity between two biological polymers, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two molecules are homologous or identical at a subunit position if both positions are occupied by the same monomer subunit, e.g., if each position in two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a linear function of the number of matched or homologous positions; e.g., if half of the positions in two sequences (e.g., five positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0088] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or a significant portion of the FR regions being those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0089] As used herein, the term "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, in which the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

[0090] As used herein, the term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is. An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-native environment, such as a host cell. In the context of this disclosure, the following abbreviations for commonly occurring nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0091] The terms "cancer-associated antigen" and "tumor antigen" are used interchangeably herein and refer to molecules (typically proteins, carbohydrates, or lipids) that are expressed either entirely or as fragments (e.g., MHC / peptides) on the surface of cancer cells and that are useful for preferential targeting of pharmacological agents to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more compared to normal cells. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, e.g., a molecule that contains deletions, additions, or mutations compared to the molecule expressed on normal cells. In some embodiments, a tumor antigen is expressed only on the cell surface of cancer cells, either entirely or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells. In some embodiments, the CARs of the present disclosure include CARs comprising an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pocket of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy.TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al, J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified from screening libraries such as human scFv phage display libraries.

[0092] The term "tumor-supporting antigen" or "cancer-supporting antigen" refers to a molecule (typically a protein, carbohydrate, or lipid) expressed on the surface of cells that are not themselves cancerous but that support cancer cells, for example, by promoting their proliferation or survival, e.g., resistance, to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). The tumor-supporting antigen itself need not play a role in supporting tumor cells, as long as the antigen is present on a cell that supports cancer cells.

[0093] As used herein in the context of scFvs, the term "flexible polypeptide linker" or "linker" refers to a peptide linker consisting of amino acids such as glycine and / or serine residues, used alone or in combination, that links the variable heavy and variable light chain regions together. According to some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer greater than or equal to 1. For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. According to some embodiments, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linker comprises multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser). Also included within the scope of this disclosure are the linkers described in WO2012 / 138475, which is incorporated herein by reference.

[0094] As used herein, a 5' cap (also called an RNA cap, RNA 7-methylguanosine cap, or RNA m G cap) is a modified guanine nucleotide added to the "front" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for recognition by ribosomes and protection from RNases. Capping is coupled to transcription and occurs co-transcriptionally, with each cap affecting the other. Shortly after transcription initiation, the 5' end of the synthesized mRNA is bound by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to modulate mRNA functionality, such as translational stability or efficiency.

[0095] The term "substantially purified," when referring to cells, refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some cases, a population of substantially purified cells refers to a homogenous population of cells. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their native state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0096] As used herein, the term "therapeutic" refers to treatment. A therapeutic effect is achieved by reducing, suppressing, ameliorating, or eradicating a disease state.

[0097] As used herein, the term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a tumor antigen) protein or carbohydrate present in a sample, but where the antibody or ligand does not substantially recognize or bind to other molecules or non-binding partners in the sample.

[0098] "Membrane anchor" or "membrane tethering domain," as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to a cell membrane.

[0099] As used herein, the term "nucleic acid" refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single-stranded or double-stranded form, including DNA, RNA, and hybrids thereof. DNA can be in the form of, for example, an antisense molecule, a plasmid DNA, a DNA-DNA duplex, a precondensed DNA, a PCR product, a vector (P1, PAC, BAC, YAC, artificial chromosome), an expression cassette, a chimeric sequence, chromosomal DNA, or derivatives and combinations thereof. DNA can be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a covalently closed linear DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), a single-stranded DNA (ssDNA), a doggybone™ DNA, a dumbbell-shaped DNA, a minimal immunologically defined gene expression (MIDGE) vector, a viral vector, or a non-viral vector. RNA can be in the form of small interfering RNA (siRNA), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and have similar binding properties to the reference nucleic acid. Examples of such analogs and / or modified residues include, but are not limited to, phosphorothioates, phosphorodiamidate morpholino oligomers (morpholinos), phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, locked nucleic acids (LNA™), and peptide nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0100] As used herein, the phrases "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any therapeutic modality that uses a nucleic acid as the active ingredient of a therapeutic agent to treat a disease or disorder. As used herein, these terms refer to RNA-based therapeutic agents and DNA-based therapeutic agents. Non-limiting examples of RNA-based therapeutic agents include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), or guide RNA (gRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or nonviral synthetic single-stranded DNA vectors (ssDNA), closed-end linear double-stranded DNA (ceDNA / CELiD), single-stranded DNA (ssDNA), plasmids, bacmids, DOGGYBONE™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (covalently closed linear DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). TNAs can be expressed or used as templates for gene editing or base editing.

[0101] As used herein, the term "AAV" or "adeno-associated virus" refers to a single-stranded DNA parvovirus that grows only in cells. Certain functions of AAV are provided only by co-infection with a helper virus. Thirteen serotypes of AAV have been identified. General information and reviews about AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York).

[0102] As used herein, the terms "single-stranded (ss) synthetic DNA molecule," "single-stranded (ss) synthetic AAV vector," "synthetic production of ssDNA molecules," and "synthetic production of ssAAV vectors" refer to single-stranded (ss) synthetic DNA molecules (ssDNA), single-stranded AAV vectors, and methods for their synthetic production in a completely cell-free environment. This production can involve one or more molecules in a manner that does not require replication or other propagation of the molecule by a cell or the interior of a cell or by the use of a cell extract. Synthetic production avoids contamination of the produced molecule with cellular contaminants, such as cellular proteins or nucleic acids, viral proteins or DNA, or insect proteins or DNA, and further avoids undesired cell-specific modifications of the molecule during the production process, such as methylation or glycosylation, or other post-translational modifications.

[0103] According to some embodiments, the 5' and / or 3' ends of certain ssDNA molecules contain approximately 145-nucleotide inverted terminal repeats (ITRs) or fragments thereof at both ends. The terminal 125 nucleotides in each ITR form a palindromic double-stranded T-shaped hairpin structure, with the A-A' palindrome forming the stem and two smaller palindromes, B-B' and C-C', forming the cross arms of the T. The other 20 nucleotides in the ITR remain single-stranded and are referred to as the D sequence. The D(-) sequence (also referred to herein as the "ssD(-) sequence") is at the 3' end, and the complementary D(-) sequence (also referred to herein as the "ssD(-) sequence") is at the 5' end. Upon double-stranded DNA synthesis, both the ssD(-) sequence and the ssD(+) sequence become double-stranded (ds)D(±) sequences, each of which contains a D region and a D' region. Ling et al. J Virol. 2015 Jan 15;89(2):952-61, WO2016081927A2 (incorporated herein by reference in its entirety) describe ssD(+) sequence-substituted ssAAV genomes. ssD(-) and ssD(+) have been reported to contain one or more transcription factor binding sites and are required for packaging and replication (Ling et al. J Virol. 2015 Jan 15;89(2):952-61, WO2016081927A2 (incorporated herein by reference in its entirety)).

[0104] As used herein, the term "stem-loop structure" refers to a nucleic acid structure comprising at least one double-stranded region (referred to herein as a "stem") and at least one single-stranded region (referred to herein as a "loop"). In some embodiments, the stem-loop structure is a hairpin structure. In some embodiments, the stem-loop structure comprises two or more stems and two or more loops. In some embodiments, a loop is located at the end of a stem (such that a single loop connects two strands of a double-stranded stem, e.g., similar to a hairpin structure). In some embodiments, a loop may be located between two stems (which may be referred to herein as a "bulge" or "bubble"), such that a loop connects two strands of different stems. In some embodiments, as described in more detail herein, a stem-loop structure may comprise a more complex secondary structure comprising multiple stems and multiple loops. As used herein, the term "ceDNA" refers to a closed-ended linear double-stranded (ds) duplex DNA, whether synthetic or otherwise, that does not contain a capsid for non-viral gene transfer. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are incorporated herein by reference. Certain methods for generating ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US18 / 49996, filed September 7, 2018, and International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for generating synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Patent Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference. According to some embodiments, the ceDNA comprises one or more phosphorothioate-modified nucleotides.

[0105] A "nucleotide" contains the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0106] "Base" includes purines and pyrimidines, which further include the naturally occurring compounds adenine, thymine, guanine, cytosine, uracil, inosine, and naturally occurring analogues, as well as synthetic derivatives of purines and pyrimidines, including, but not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0107] As used herein, the term "interfering RNA" or "RNAi" or "interfering RNA sequence" includes single-stranded RNA (e.g., mature miRNA, ssRNAi oligonucleotide, ssDNAi oligonucleotide), double-stranded RNA (i.e., double-stranded RNA such as siRNA, Dicer substrate dsRNA, shRNA, aiRNA, or pre-miRNA), DNA-RNA hybrid (e.g., see PCT Publication No. 2004 / 078941), or DNA-DNA hybrid (e.g., see PCT Publication No. 2004 / 104199), which can reduce or inhibit the expression of a target gene or sequence (e.g., by mediating the degradation of mRNA complementary to the interfering RNA sequence or inhibiting its translation) when the interfering RNA is present in the same cell as the target gene or sequence. Thus, interfering RNA refers to a single-stranded RNA complementary to a target mRNA sequence, or a double-stranded RNA formed by two complementary strands or a single self-complementary strand. The interfering RNA may have substantial or complete identity with the target gene or sequence, or may contain mismatch regions (i.e., mismatch motifs). The sequence of the interfering RNA may correspond to the full-length target gene or a subsequence thereof. Preferably, the interfering RNA molecule is chemically synthesized. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0108] Interfering RNA includes "small interfering RNA" or "siRNA," e.g., an interfering RNA that is about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary strand of a double-stranded siRNA). The target sequence is 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 nucleotides in length, preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides in length, and the double-stranded siRNA is about 15 to 60, 15 to 50, 15 to 40, 15 to 30, 15 to 25, or 19 to 25 base pairs in length, preferably about 18 to 22, 19 to 20, or 19 to 21 base pairs in length.) The siRNA duplex may comprise a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include but are not limited to: double-stranded polynucleotide molecules that are assembled from two separate chain-like molecules, one strand being sense strand and the other being complementary antisense strand; double-stranded polynucleotide molecules that are assembled from single-stranded molecules that sense region and antisense region are linked by nucleic acid-based linker or non-nucleic acid-based linker; double-stranded polynucleotide molecules that have hairpin secondary structure with self-complementary sense region and antisense region; and circular single-stranded polynucleotide molecules that have stem with two or more loop structures and self-complementary sense region and antisense region, and circular polynucleotide can be processed in vivo or in vitro to produce active double-stranded siRNA molecules.As used herein, the term " siRNA " includes RNA-RNA duplex and DNA-RNA hybrid (see, for example, PCT Publication No. 2004 / 078941).

[0109] The phrase "immunosuppressants" refers to a group of small molecule, monoclonal antibody, or polypeptide antagonists that inhibit protein kinases, such as tyrosine kinases. The term immunosuppressant also includes any agent, including antibodies and other protein drugs, that inhibits or blocks the activity of the immune system, for example, in the case of allergic reactions, inflammatory or autoimmune disorders, transplant rejection, or graft-versus-host disease.

[0110] As used herein, the term "tyrosine kinase inhibitor" or "TKI" refers to a molecule that inhibits tyrosine kinase activity. A tyrosine kinase inhibitor can be, for example, a small molecule inhibitor, a biologic (such as a monoclonal antibody), or a large polypeptide molecule that inhibits, for example, the activity of the IFN signaling and production pathway, or any other form of antagonist that can reduce the expression or activity of a tyrosine kinase.

[0111] The phrases "anti-therapeutic nucleic acid immune response," "anti-transfer vector immune response," "immune response to a therapeutic nucleic acid," "immune response to a transfer vector," and the like refer to any unwanted immune response to a therapeutic nucleic acid, whether of viral or non-viral origin. In some embodiments, the unwanted immune response is an antigen-specific immune response against the viral transfer vector itself. In some embodiments, the immune response is specific to the transfer vector, which may be double-stranded DNA, single-stranded DNA, single-stranded RNA, or double-stranded RNA. In other embodiments, the immune response is specific to the sequence of the transfer vector. In other embodiments, the immune response is specific to the CpG content of the transfer vector.

[0112] "Decrease," "decreasing," "reduce," or "reducing" an immune response by an immunosuppressant is intended to mean a detectable decrease in the immune response to a given immunosuppressant. The amount of decrease in the immune response by an immunosuppressant can be determined relative to the level of the immune response in the presence of the immunosuppressant. A detectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more below the immune response detected in the presence of the immunosuppressant. A reduced immune response in the presence of an immunosuppressant is typically measured by a reduction in cytokine production (e.g., IFNα, IFNγ, TNFα, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, or IL-18) by responder cells in vitro or in the serum of a mammalian subject following administration of an interfering RNA.

[0113] As used herein, the term "responder cell" refers to a cell, preferably a mammalian cell, that produces a detectable immune response when contacted with an immunostimulatory therapeutic nucleic acid. Exemplary responder cells include, for example, dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Exemplary responder cells can be human THP1 monocytes and murine RAW macrophage cells. Detectable immune responses can be readily measured in vitro, for example, using THP1-interferon-stimulated gene (ISG) or RAW-ISG cells with various reporter constructs, including interferon regulatory factor (IRF)-inducible reporter constructs. In vivo immune responses can be measured by determining the production levels of cytokines or growth factors, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-18, IP-10, TGF, VEGF, VEGFR, or combinations thereof. Additionally, immune responses can also be measured by detecting the levels of chemokines, such as MCP-1, MIP-1α (CCL3), MIP-1β (CCL4), and Rantes (CCL5).

[0114] The term "lipid" refers to a group of organic compounds, including, but not limited to, esters of fatty acids, characterized by being insoluble in water but soluble in many organic solvents. Lipids are typically divided into at least three classes: (1) "simple lipids," which include fats and oils as well as waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0115] As used herein, the terms "lipid particle" or "lipid nanoparticle" (LNP) refer to lipid formulations that can be used to deliver therapeutic agents, such as therapeutic nucleic acids, to desired target sites (e.g., cells, tissues, organs, etc.). In some embodiments, the lipid nanoparticles of the present disclosure are typically formed from ionizable lipids (e.g., cationic lipids), sterols (e.g., cholesterol), conjugate lipids (e.g., lipid-anchored polymers) that prevent particle aggregation, and optionally helper lipids (e.g., non-cationic lipids). In some other embodiments, a therapeutic agent, such as a therapeutic nucleic acid (TNA), may be encapsulated in the lipid particle, thereby protecting it from degradation. In yet other embodiments, an immunosuppressant may optionally be included in the nucleic acid comprising the lipid nanoparticle. In one embodiment, the lipid particle comprises a nucleic acid (e.g., ceDNA, ssDNA, and / or mRNA). The present disclosure provides LNPs in which at least one of the lipids in the lipid-anchored polymer contains 16, 18, or 20 aliphatic carbons to more firmly anchor the lipid-anchored polymer to the LNP. In some embodiments, at least one lipid in a lipid-anchored polymer having at least 18 aliphatic carbons is useful for generating stealth LNPs. In other embodiments, at least one lipid in a lipid-anchored polymer having at least 20 aliphatic carbons is useful for generating stealth LNPs.

[0116] According to some embodiments, lipid particles of the present disclosure typically have a size of about 20 nm to about 90 nm, about 25 nm to about 80 nm, about 25 nm to about 75 nm, about 25 nm to about 70 nm, about 30 nm to about 75 nm, about 30 nm to about 70 nm, about 35 nm to about 75 nm, about 35 nm to about 70 nm, about 40 nm to about 75 nm, about 40 nm to about 70 nm, about 45 nm to about 75 nm, about 50 nm to about 55 nm, about 55 nm to about 50 ... 0nm to about 75nm, about 50nm to about 70nm, about 60nm to about 75nm, about 60nm to about 70nm, about 65nm to about 75nm, about 65nm to about 70nm, or about 20nm, about 25nm m, approximately 30nm, approximately 35nm, approximately 40nm, approximately 45nm, approximately 50nm, approximately 51nm, approximately 52nm, approximately 53nm, approximately 54nm, approximately 55nm, approximately 56nm, approximately 57nm, approximately 58nm, approximately 59 nm has an average diameter of a size of about 60 nm, about 61 nm, about 62 nm, about 63 nm, about 64 nm, about 65 nm, about 66 nm, about 67 nm, about 68 nm, about 69 nm, about 70 nm, about 71 nm, about 72 nm, about 73 nm, about 74 nm, or about 75 nm (±3 nm).

[0117] Generally, the LNPs of the present disclosure have an average diameter selected to produce an intended therapeutic effect. For example, the LNPs of the present disclosure have an average diameter that is compatible with a target organ (e.g., the liver) so that the LNPs of the present disclosure can diffuse through fenestrations in the target organ (e.g., liver) or target cell subpopulation (e.g., hepatocytes).

[0118] According to some embodiments, the lipid particles of the present disclosure typically have an average diameter of less than about 100 nm, less than about 90 nm, less than about 80 nm, less than about 75 nm, less than about 70 nm, less than about 65 nm, less than about 60 nm, less than about 55 nm, less than about 50 nm, less than about 45 nm, less than about 40 nm, less than about 35 nm, less than about 30 nm, less than about 25 nm, or less than about 20 nm in size.

[0119] As used herein, the term "cationic lipid" refers to any lipid that is positively charged at physiological pH.The cationic lipid in lipid particles can include one or more cationic lipids, such as, for example, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), "SS-cleavable lipid", or a mixture thereof.In some embodiments, the cationic lipid can also be an ionizable lipid, i.e., an ionizable cationic lipid. The term "cationic lipid" also encompasses lipids that are positively charged at any pH, such as lipids containing quaternary amine groups, i.e., quaternary lipids. Any cationic lipid described herein that contains a primary, secondary, or tertiary amine group can be converted to the corresponding quaternary lipid, for example, by treatment with a solution of chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl).

[0120] As used herein, the term "ionizable lipid" is intended to refer to a lipid, e.g., a cationic lipid, that has at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. It will be understood by those skilled in the art that the addition or removal of protons as a function of pH is an equilibrium process, and reference to a charged or neutral lipid refers to the nature of the predominant species, and does not require that all lipids exist in a charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. In some embodiments, ionizable lipids may include "cleavable lipids" or "SS-cleavable lipids."

[0121] As used herein, the term "neutral lipid" is intended to refer to any of several lipid species that exist in either an uncharged form or a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0122] As used herein, the term "non-cationic lipid" is intended to refer to any amphipathic lipid, as well as any other neutral or anionic lipid.

[0123] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to an ionizable lipid containing a disulfide bond-cleavable unit. The cleavable lipid may contain a cleavable disulfide bond (SS) containing pH-sensitive amine, e.g., a tertiary amine, and a self-degrading phenyl ester-containing lipid-like material. For example, the SS-cleavable lipid can be ss-OP lipid (COATSOME® SS-OP), ss-M lipid (COATSOME® SS-M), ss-E lipid (COATSOME® SS-E), ss-EC lipid (COATSOME® SS-EC), ss-LC lipid (COATSOME® SS-LC), ss-OC lipid (COATSOME® SS-OC), and ss-PalmE lipid (see, e.g., Formulas I-IV), or lipids described in Togashi et al., (2018) Journal of Controlled Release “A hepatic pDNA delivery system based on an intracellular environment sensitive vitamin E-scaffold lipid-like material with the aid of an anti-inflammatory drug” 279:262-270. Further examples of cleavable lipids are described in U.S. Patent No. 9,708,628 and U.S. Patent No. 10,385,030, the entire contents of which are incorporated herein by reference. In one embodiment, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds, which can be cleaved in a reducing environment, such as an acidic compartment, for example, an endosome or lysosome for membrane destabilization, and the cytoplasm. In one embodiment, the cleavable lipid is a cationic lipid. In one embodiment, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0124] As used herein, "encapsulated lipid" refers to a lipid nanoparticle that provides an active or therapeutic agent, such as a nucleic acid (e.g., ceDNA, non-viral ssDNA, or mRNA), with complete encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is completely encapsulated in the lipid nanoparticle (e.g., to form a lipid nanoparticle that encapsulates the nucleic acid).

[0125] The term "lipid-anchored polymer" or "lipid polymer" or "lipid conjugate" refers to a conjugated lipid that inhibits lipid particle aggregation. Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as PEG conjugated to DSG (e.g., PEG-DSG conjugate), PEG conjugated to DSPE (e.g., PEG-DSPE conjugate), and PEG conjugated to ceramide (see, e.g., U.S. Pat. No. 5,885,613), polyglycerol (PG)-lipid conjugates such as DODA-PG, and mixtures thereof. An example of a PG-lipid conjugate is DODA-PG45. Further examples of POZ-lipid conjugates are described in PCT Publication No. 2010 / 006282. PEG, PG, or POZ can be directly conjugated to the lipid or can be linked to the lipid via a linker moiety. For example, any linker moiety suitable for linking PEG, PG, or POZ to the lipid can be used, including non-ester-containing and ester-containing linker moieties. In certain preferred embodiments, non-ester containing linker moieties such as amides or carbamates are used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes.

[0126] As used herein, the term "lipid-anchored polymer," which may be used interchangeably with the terms "lipid conjugate" or "lipid polymer," refers to a molecule comprising a lipid moiety covalently attached to a hydrophilic polymer, optionally via a linker. Without wishing to be bound by any particular theory, it is believed that the lipid-anchored polymer inhibits aggregation of LNPs and provides in vivo steric stabilization and a prolonged blood half-life (t 1 / 2Lipid moieties ("lipid linkers" or "linker lipids") in which the linker is conjugated to a hydrophilic polymer (e.g., PEG, PG, or POZ) include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylinositol (DSPE), 1,2-diphenylphosphine-3-phosphate (PD ... 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In one embodiment, the lipid-anchored polymer comprises a linker lipid moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof, as well as any combination of the foregoing. For example, PEG2000 bound to DSG is the lipid-anchored polymer PEG2000-DSG (or DSG-PEG2000). PEG bound to DSPE is the lipid-anchored polymer PEG-DSPE (or DSPE-PEG2000 or DSPE-PEG500).An example of a lipid-anchored PG polymer can include DODA-PG, where the PG can be a multi-unit ranging from about 5 to about 50 PG units.

[0127] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine.Other compounds that lack phosphorus, such as sphingolipids, glycosphingolipid family, diacylglycerol, and β-acyloxyacid, are also included in the group called amphipathic lipid.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0128] The term "neutral lipid" refers to any of several lipid species that exist in either an uncharged form or a neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0129] The term "non-cationic lipid" or "helper lipid" refers to any amphipathic lipid, as well as any other neutral or anionic lipid, including, but not limited to, distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0130] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH, including, but not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleylphosphatidylglycerol (POPG), and other anionic modifying groups linked to neutral lipids.

[0131] The term "hydrophobic lipid" refers to a compound having a non-polar group, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and groups optionally substituted with one or more aromatic, alicyclic, or heterocyclic groups. Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N-N-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0132] As used herein, the term "aqueous solution" refers to a composition that comprises, in whole or in part, water.

[0133] As used herein, the term "organic lipid solution" refers to a composition that comprises, in whole or in part, an organic solvent with a lipid.

[0134] As used herein, "systemic delivery" refers to the delivery of lipid particles that results in widespread biodistribution of an active agent (e.g., CAR T) within an organism. Some administration techniques can result in systemic delivery of a particular agent, while others cannot. Systemic delivery means that a useful amount, preferably a therapeutic amount, of the agent is exposed to most parts of the body. To achieve widespread biodistribution, a blood lifetime is generally required such that the agent is not rapidly degraded or excreted (e.g., by first-pass organs (liver, lung, etc.) or rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, the systemic delivery of lipid particles is by intravenous delivery.

[0135] As used herein, the term "off-target delivery" refers to the delivery of the LNPs of the present disclosure to non-target cells. After administration to a subject, the LNPs may be delivered to the non-target cells, resulting in expression of the therapeutic nucleic acid (TNAs) in the non-target cells.

[0136] As used herein, "local delivery" refers to the delivery of an active agent, such as ceDNA, ssDNA, mRNA, or interfering RNA (e.g., siRNA), to a target site within an organism. For example, the agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as a site of inflammation, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0137] As used herein, the term "ceDNA" refers to closed-ended, linear, double-stranded (ds) duplex DNA, whether synthetic or otherwise, that does not contain a capsid for non-viral gene transfer. A detailed description of ceDNA is provided in International Patent Application No. PCT / US2017 / 020828, filed March 3, 2017, the entire contents of which are expressly incorporated herein by reference. Certain methods for producing ceDNA containing various inverted terminal repeat (ITR) sequences and configurations using cell-based methods are described in Example 1 of International Patent Application No. PCT / US18 / 49996, filed September 7, 2018, and International Patent Application No. PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for the generation of synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International Patent Application No. PCT / US2019 / 14122, filed January 18, 2019, the entire contents of which are incorporated herein by reference. As used herein, the terms "ceDNA vector" and "ceDNA" are used interchangeably.

[0138] As used herein, the term "neDNA" or "nicked ceDNA" refers to closed-end DNA that has a nick or gap of 1 to 100 base pairs in the stem or spacer region 5' upstream of the open reading frame (e.g., the promoter and transgene to be expressed).

[0139] As used herein, the terms "gap" and "nick" are used interchangeably and refer to an interrupted portion of the synthetic DNA vector of the present disclosure that creates a continuous single-stranded DNA segment in an otherwise double-stranded ceDNA. A gap can be 1 nucleotide (nt) to 100 nucleotides (nt) in length in one strand of the duplex DNA. Exemplary gaps designed and generated by the methods described herein, and synthetic vectors produced thereby, can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 bp in length. Exemplary gaps in the present disclosure can be 1 nt to 10 nt in length, 1 to 20 nt in length, or 1 to 30 nt in length.

[0140] As used herein, the term "inverted terminal repeat" or "ITR" is intended to refer to a nucleic acid sequence located at the 5' and / or 3' end of an ssDNA vector disclosed herein, comprising at least one stem-loop structure that is partially duplexed and includes at least one loop. According to some embodiments, the ITR may be an artificial sequence (e.g., does not contain sequences derived from a virus). The ITR may further comprise one stem-loop structure (e.g., a "hairpin") or two or more stem-loop structures. For example, the ITR may comprise two stem-loop structures (e.g., a "hammerhead," "dogbone," or "dumbbell"), three stem-loop structures (e.g., a "cruciform"), or a more complex structure. The ITR may comprise an aptamer sequence or one or more chemical modifications.

[0141] According to some embodiments, "ITRs" can be artificially synthesized using a set of oligonucleotides containing one or more desired functional sequences (e.g., palindromic sequences). The ITR sequences can be artificial AAV ITRs, artificial non-AAV ITRs, or ITRs physically derived from viral AAV ITRs (e.g., ITR fragments removed from the viral genome). For example, ITRs can be derived from the Parvoviridae family, which includes parvoviruses and dependoviruses (e.g., canine parvovirus, bovine parvovirus, mouse parvovirus, porcine parvovirus, and human parvovirus B-19), or the SV40 hairpin that functions as the origin of SV40 replication can be used as an ITR, which can be further modified by truncation, substitution, deletion, insertion, and / or addition. The Parvoviridae family of viruses consists of two subfamilies: Parvovirinae, which infect vertebrates, and Densovirinae, which infect invertebrates. Dependoparvoviruses include the adeno-associated virus (AAV) family of viruses that can replicate in vertebrate hosts, including, but not limited to, humans, primates, bovine, canine, equine, and bovine species. Typically, ITR sequences can be derived from AAV, as well as parvoviruses, lentiviruses, goose viruses, and B19, in wild-type, "dogbone," and "dumbbell" configurations, symmetric, or even asymmetric ITR orientations. While ITRs are typically present at both the 5' and 3' ends of AAV vectors in single-stranded DNA (ssDNA) molecules, ITRs can be present at only one end of a linear vector. For example, ITRs can be present only at the 5' end. In some other cases, ITRs can be present only at the 3' end in single-stranded DNA (ssDNA) molecules. For convenience herein, an ITR located 5' to (upstream of) an expression cassette of a single-stranded DNA (ssDNA) molecule vector is referred to as the "5' ITR" or "left ITR," and an ITR located 3' to (downstream of) an expression cassette of a single-stranded DNA (ssDNA) molecule is referred to as the "3' ITR" or "right ITR."

[0142] As used herein, "wild-type ITR" or "WT-ITR" refers to the sequence of a naturally occurring ITR sequence in an AAV or other dependovirus that retains, for example, Rep binding activity and Rep nicking ability. The nucleic acid sequence of a WT-ITR from any AAV serotype may differ slightly from the naturally occurring reference sequence due to degeneracy or drift in the genetic code; therefore, WT-ITR sequences encompassed for use herein include WT-ITR sequences that result from naturally occurring variations (e.g., replication errors).

[0143] As used herein, the terms "substantially symmetric WT-ITR" or "substantially symmetric WT-ITR pair" refer to a pair of wild-type ITRs in a synthetic AAV vector, both of which have reverse-complementary sequences throughout their entire length. For example, an ITR can be considered a wild-type sequence even if it has one or more nucleotides that deviate from a naturally occurring reference sequence, as long as the changes do not affect the physical and functional properties and overall three-dimensional structure (secondary and tertiary) of the sequence. In some embodiments, the deviating nucleotides represent conservative sequence changes. As one non-limiting example, a sequence that has at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (e.g., as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial configuration relative to the other WT-ITR, such that their three-dimensional structures have the same shape in geometric space. A substantially symmetric WT-ITR has the same A, C-C', and B-B' loops in three-dimensional space. A substantially symmetric WT ITR can be functionally confirmed as WT by determining that it has an operable Rep binding site (RBE or RBE') and terminal resolution site (TRS) that pairs with the appropriate Rep protein. One skilled in the art can optionally test for other functions, including transgene expression under permissive conditions.

[0144] As used herein, the phrases "modified ITR" or "mod-ITR" or "mutated ITR" are used interchangeably herein and refer to an ITR that has a mutation in at least one nucleotide compared to a wild-type ITR from the same serotype. The mutation can result in a change in one or more of the A, C, C', B, and B' regions in the ITR, and can result in a change in the three-dimensional spatial configuration (i.e., its three-dimensional structure in geometric space) compared to the three-dimensional spatial configuration of a wild-type ITR from the same serotype.

[0145] As used herein, the term "asymmetric ITR," also referred to as an "asymmetric ITR pair," refers to a pair of ITRs in a single-stranded synthetic AAV genome that are not reverse complements over their entire length. As one non-limiting example, an asymmetric ITR pair does not have a symmetrical three-dimensional spatial configuration relative to its cognate ITR, such that their three-dimensional structures are different shapes in geometric space. In other words, an asymmetric ITR pair has a different overall geometric structure, i.e., they have different configurations of their A, C-C', and B-B' loops in three-dimensional space (e.g., one ITR may have a shorter C-C' arm and / or a shorter B-B' arm compared to the cognate ITR). The sequence difference between the two ITRs may be due to one or more nucleotide additions, deletions, truncations, or point mutations. According to some embodiments, one ITR of the asymmetric ITR pair may be a wild-type AAV ITR sequence, and the other ITR may be a modified ITR (e.g., a non-wild-type or synthetic ITR sequence) as defined herein. In another embodiment, neither ITR of the asymmetric ITR pair is a wild-type AAV sequence, but rather the two ITRs are modified ITRs with different shapes in geometric space (i.e., different overall geometric structures). In some embodiments, one mod-ITR of the asymmetric ITR pair can have a short C-C' arm, and the other ITR can have a different modification (e.g., a single arm or a short B-B' arm) such that it has a different three-dimensional spatial configuration compared to the cognate asymmetric mod-ITR.

[0146] As used herein, the term "symmetric ITRs" refers to a pair of ITRs in a single-stranded AAV genome that are mutated or modified compared to the wild-type depend virus ITR sequences and are reverse-complementary across their entire length. Neither ITR is the wild-type ITR AAV2 sequence (i.e., they are modified ITRs, also referred to as mutant ITRs), and may have a sequence that differs from the wild-type ITR due to nucleotide additions, deletions, substitutions, truncations, or point mutations. For convenience herein, the ITR located 5' to (upstream of) the expression cassette of a synthetic AAV vector is referred to as the "5' ITR" or "left ITR," and the ITR located 3' to (downstream of) the expression cassette of a synthetic AAV vector is referred to as the "3' ITR" or "right ITR."

[0147] As used herein, the term "substantially symmetric modified ITR" or "substantially symmetric mod-ITR pair" refers to a pair of modified ITRs in a synthetic AAV that both have reverse-complementary sequences throughout their entire length. For example, modified ITRs can be considered substantially symmetric even if they have some nucleotide sequence deviations from their reverse-complementary sequences, as long as the changes do not affect their properties and overall shape. As one non-limiting example, a sequence that has at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a reference sequence (as measured using BLAST with default settings) and also has a symmetrical three-dimensional spatial organization relative to its cognate modified ITRs such that their three-dimensional structures have the same shape in geometric space. In other words, a substantially symmetric modified ITR pair has the same A, C-C', and B-B' loops organized in three-dimensional space. In some embodiments, the ITRs from a mod-ITR pair can have different reverse-complementary nucleotide sequences but still have the same symmetrical three-dimensional spatial organization, i.e., both ITRs have mutations that result in the same overall three-dimensional shape. For example, one ITR (e.g., the 5' ITR) of a mod-ITR pair can be from one serotype, and the other ITR (e.g., the 3' ITR) can be from a different serotype, but both can have the same corresponding mutations such that the modified ITR pair has the same symmetrical three-dimensional spatial organization (e.g., if the 5' ITR has a deletion in the C region, the cognate modified 3' ITR from the different serotype has a deletion at a corresponding position in the C' region). In such embodiments, each ITR of a modified ITR pair can be from a different serotype (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), e.g., a combination of AAV2 and AAV6, with modifications in one ITR mirrored at the corresponding position in the cognate ITR from the different serotype. According to some embodiments, a substantially symmetric modified ITR pair refers to a pair of modified ITRs (mod-ITRs) so long as the nucleotide sequence differences between the ITRs do not affect their properties or overall shape and they have substantially the same shape in three-dimensional space.Non-limiting examples include mod-ITRs that have at least 95%, 96%, 97%, 98%, or 99% sequence identity with a reference mod-ITR, as determined by standard means known in the art, such as BLAST (Basic Local Alignment Search Tool) or BLASTN with default settings, and also have symmetrical three-dimensional spatial configurations such that their three-dimensional structures are the same shape in geometric space. A substantially symmetric mod-ITR pair will have the same A, C-C', and B-B' loops in three-dimensional space; for example, if the modified ITR of a substantially symmetric mod-ITR pair has a deletion of the C-C' arm, the cognate mod-ITR will have a corresponding deletion of the C-C' loop and also have a similar three-dimensional structure of the remaining A and B-B' loops that are the same shape in the geometric space of the cognate mod-ITR.

[0148] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence with respect to another. Generally, in the sequence ABC, B is flanked by A and C. Similarly for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the flanked sequence, but need not be contiguous with or immediately adjacent to it. According to some embodiments, the term flanking refers to the terminal repeat sequences at each end of a linear, single-stranded synthetic AAV vector.

[0149] As used herein, the term "spacer region" refers to an intervening sequence that separates functional elements within a vector or genome. In some embodiments, an AAV spacer region maintains two functional elements at a desired distance for optimal functionality. In some embodiments, the spacer region provides or adds to the genetic stability of the vector or genome. In some embodiments, the spacer region facilitates easy genetic manipulation of the genome by providing conveniently located cloning sites and gaps of a designed number of base pairs. For example, in certain aspects, oligonucleotide "polylinkers" or "polycloning sites" containing several restriction endonuclease sites, or non-open reading frame sequences designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in the vector or genome to separate cis-acting elements, similar to an AAV vector or genome, for example, by inserting a 6-mer, 12-mer, 18-mer, 24-mer, 48-mer, 86-mer, 176-mer, etc., between the terminal resolution site and the upstream transcriptional regulatory element.

[0150] As used herein, the terms "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and refer to a binding site for a Rep protein (e.g., AAV Rep 78 or AAV Rep 68) that, upon binding by the Rep protein, enables the Rep protein to perform its site-specific endonuclease activity on a sequence incorporating the RBS. An RBS sequence and its reverse complement together form a single RBS. RBS sequences are well known in the art and include, for example, the RBS sequence specified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'.

[0151] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and refer to the region where Rep forms a tyrosine-phosphodiester bond with 5' thymidine, generating a 3'-OH that serves as a substrate for DNA elongation via a cellular DNA polymerase, e.g., DNA pol delta or DNA pol epsilon. Alternatively, the Rep-thymidine complex may participate in a coordinate ligation reaction.

[0152] As used herein, the terms "sense" and "antisense" refer to the orientation of structural elements on a polynucleotide. The sense and antisense versions of an element are the reverse complements of each other.

[0153] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" refer to AAV vectors and methods for their synthetic production in a completely cell-free environment.

[0154] As defined herein, a "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally result in a measurable signal, such as fluorescence, color, or luminescence. A reporter protein coding sequence encodes a protein whose presence in a cell or organism is easily observed.

[0155] As used herein, the term "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the composition. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxic, allergic, or similar untoward reactions when administered to a host.

[0156] As used herein, the term "in vivo" refers to an assay or process performed on or within an organism, such as a multicellular animal. In some of the aspects described herein, a method or use can be said to be performed "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses performed using living cells with intact membranes outside a multicellular animal or plant, for example, explants, cultured cells including primary cells and cell lines, transformed cell lines, and extracted tissues or cells including blood cells, among others. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and can refer to the introduction of a programmable synthetic biological circuit in a non-cellular system, such as a cell- or cell-line-free medium, such as a cell extract.

[0157] As used herein, the term "promoter" refers to any nucleic acid sequence that regulates the expression of another nucleic acid sequence by driving the transcription of that nucleic acid sequence, which may be a heterologous target gene encoding a protein or RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. Promoters can also contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Within the promoter sequence, transcription initiation sites and protein binding domains involved in the binding of RNA polymerase are found. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive transgene expression in the synthetic AAV vectors disclosed herein. The promoter sequence can be bounded at its 3' end by a transcription initiation site and can extend upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background.

[0158] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and refer to a heterologous DNA sequence operably linked to a promoter or other DNA regulatory sequence sufficient to direct transcription of a transgene in a DNA vector, e.g., a synthetic AAV vector. Suitable promoters include, for example, tissue-specific promoters. The promoter can also be of AAV origin.

[0159] As used herein, "operably linked" refers to a juxtaposition in which the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. A promoter can be said to drive expression or drive the transcription of a nucleic acid sequence that it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" refer to a promoter in the correct functional location and / or orientation with respect to the nucleic acid sequence it regulates so as to control transcription initiation and / or expression of the nucleic acid sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in an inverted orientation, such that what was the coding strand is now the non-coding strand (or vice versa). Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0160] The terms "DNA regulatory sequence," "control element," and "regulatory element," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, etc., that provide and / or regulate the transcription of a non-coding sequence (e.g., a DNA-targeting RNA) or a coding sequence (e.g., a site-directed modifying polypeptide or a Cas9 / Csn1 polypeptide) and / or regulate the translation of an encoded polypeptide.

[0161] A promoter can be a promoter naturally associated with a gene or sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exons of a given gene or sequence. Such a promoter can be referred to as "endogenous." Similarly, according to some embodiments, an enhancer can be an enhancer naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments, a coding nucleic acid segment is positioned under the control of a "recombinant promoter" or a "heterologous promoter," both of which refer to a promoter that is not normally associated with the coding nucleic acid sequence to which it is operably linked in its natural environment. Similarly, a "recombinant or heterologous enhancer" refers to an enhancer that is not normally associated with a given nucleic acid sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, as well as synthetic promoters or enhancers that are not "naturally occurring," i.e., contain different elements of different transcriptional regulatory regions and / or mutations that alter expression using genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be generated using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in conjunction with the synthetic biological circuits and modules disclosed herein (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906, each of which is incorporated herein by reference). Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences within non-nuclear organelles, such as mitochondria and chloroplasts, can also be used.

[0162] As described herein, an "inducible promoter" is characterized by initiating or enhancing transcriptional activity when affected by or contacted with an inducer or inducing agent. As defined herein, an "inducer" or "inducing agent" can be an endogenous or, usually, exogenous compound or protein that is administered in such a way that it is active in inducing transcriptional activity from an inducible promoter. In some embodiments, the inducer or inducing agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., an inducer can be an inducer protein expressed by another component or module), which can itself be under the control of an inducible promoter. In some embodiments, an inducible promoter is induced in the absence of certain agents, such as a repressor. Examples of inducible promoters include, but are not limited to, tetracycline, metallothionine, ecdysone, mammalian viruses (e.g., adenovirus late promoter and mouse mammary tumor virus long terminal repeat (MMTV-LTR)), as well as other steroid-responsive promoters, rapamycin-responsive promoters, and the like.

[0163] As used herein, the term "subject" is intended to include a living organism (e.g., a mammal, a human) in which an immune response can be elicited. Typically, animals are vertebrates, such as, but not limited to, primates, rodents, domestic animals, or game animals. Primates include, but are not limited to, chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include, but are not limited to, cattle, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cats, canine species, e.g., dogs, foxes, wolves, avian species, e.g., chickens, emus, ostriches, and fish, e.g., trout, catfish, and salmon. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate or a human. The subject can be male or female. Additionally, the subject can be an infant or a child. In some embodiments, the subject can be a newborn or unborn subject, e.g., the subject is present in utero. Preferably, the subject is a mammal. The mammal can be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects that represent animal models of diseases and disorders. Additionally, the methods and compositions described herein can be used with domestic animals and / or pets. Human subjects can be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Hispanic, Middle Eastern, etc. In some embodiments, the subject can be a patient or other subject in a clinical setting. In some embodiments, the subject has already received treatment. In some embodiments, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult. In some embodiments, the subject is a human fetus, human newborn, human infant, human child, human adolescent, or human adult. In some embodiments, the subject is an animal embryo, or a non-human embryo, or a non-human primate embryo.In some embodiments, the subject is a human embryo.

[0164] As used herein, the term "therapeutic" means treatment. A therapeutic effect is achieved by reducing, suppressing, ameliorating, or eradicating a disease state.

[0165] As used herein, the term "prophylaxis" refers to the prevention or protective treatment of a disease or condition. In the context of the present disclosure, a "tumor antigen," or a "hyperproliferative disorder antigen," or an "antigen associated with a hyperproliferative disorder" refers to an antigen common to a particular hyperproliferative disorder. In certain aspects, the hyperproliferative disorder antigens of the present disclosure are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and adenocarcinomas, such as breast cancer, prostate cancer, ovarian cancer, and pancreatic cancer.

[0166] As used herein, the term "host cell" includes any cell type amenable to transformation, transfection, transduction, etc. with a nucleic acid therapeutic of the present disclosure. By way of non-limiting example, a host cell can be an immunostimulatory cell such as a T cell, a B cell, a dendritic cell, or a natural killer (NK) cell.

[0167] As used herein, the term "exogenous" refers to a substance present in a cell other than its natural source. As used herein, the term "exogenous" can refer to a nucleic acid (e.g., a nucleic acid encoding a polypeptide) or polypeptide that has been introduced by a process requiring human intervention into a biological system, such as a cell or organism, where it is not normally found and it is desired to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, "exogenous" can refer to a nucleic acid or polypeptide that has been introduced by a process requiring human intervention into a biological system, such as a cell or organism, where it is found in relatively low amounts and it is desired to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to result in ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0168] As used herein, the term "sequence identity" refers to the relationship between two nucleotide sequences. For purposes of this disclosure, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Needle program in the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al. et al., 2000, supra), preferably using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) implemented in version 3.0.0 or later. Optional parameters used are a gap opening penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version in NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity, calculated as follows: (identical deoxyribonucleotides × 100) / (length of alignment - total number of gaps in the alignment). The length of the alignment is preferably at least 10 nucleotides, preferably at least 25 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides.

[0169] As used herein, the term "homology" or "homology" is defined as the percentage of nucleotide residues in the homologous arm that are identical to the nucleotide residues in the corresponding sequence on the target chromosome after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage. Alignment for determining the percentage of nucleotide sequence homology can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. In some embodiments, for example, a nucleic acid sequence (e.g., a DNA sequence) of a homologous arm of a repair template is considered to be "homologous" if the sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to the corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of the host cell.

[0170] As used herein, the term "heterologous," as used herein, refers to a nucleotide sequence or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a naturally occurring nucleic acid sequence (or a variant thereof) to generate a chimeric nucleotide sequence that encodes a chimeric polypeptide. A heterologous nucleic acid sequence can be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleic acid sequence that encodes a fusion variant polypeptide.

[0171] As used herein, a "vector" or "expression vector" is a replicon, such as a plasmid, bacmid, phage, virus, virion, or cosmid, to which another DNA segment, i.e., an "insert," "transgene," or "expression cassette," can be attached so as to bring about expression or replication of the attached segment ("expression cassette") in a cell. A vector can be a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be of viral or non-viral origin in its final form. However, for the purposes of this disclosure, "vector" generally refers to a synthetic AAV vector or a nicked ceDNA vector. Thus, the term "vector" encompasses any genetic element that, when associated with the appropriate control elements, is capable of replication and of transferring gene sequences to a cell. In some embodiments, a vector can be a recombinant vector or an expression vector.

[0172] As used herein, the phrase "recombinant vector" refers to a vector containing a heterologous nucleic acid sequence or "transgene" that can be expressed in vivo. It is understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a public episomal vector provides a means of maintaining a nucleotide of interest in a subject in high copy number extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0173] As used herein, the term "expression" refers to the cellular processes involved in the production of RNA and proteins, and optionally secreted proteins, including, for example, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. As used herein, the phrase "expression product" includes RNA transcribed from a gene (e.g., a transgene) and polypeptides obtained by translation of mRNA transcribed from a gene.

[0174] As used herein, the term "gene" means a nucleic acid sequence that is transcribed (from DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.

[0175] Pharmacokinetic principles provide a basis for modifying dosing regimens to achieve a desired degree of therapeutic efficacy with minimal unacceptable adverse effects. Additional guidance for dosage modifications can be obtained in situations where the plasma concentration of the drug is measured and can be related to a therapeutic window.

[0176] As used herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms to achieve a beneficial or desired clinical result. Treating further refers to achieving one or more of: (a) reducing the severity of the disorder; (b) limiting the onset of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and (e) limiting the recurrence of symptoms in patients who did not previously exhibit symptoms of the disorder. In one aspect of any of the aspects or embodiments herein, the terms "treat," "treating," and / or "treatment" include arresting, inhibiting, slowing, or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.

[0177] Beneficial or desired clinical results, e.g., pharmacological and / or physiological effects, include, but are not limited to, preventing a disease, disorder, or condition from occurring in a subject who may be predisposed to the disease, disorder, or condition but who has not yet experienced or exhibited symptoms of the disease (prophylactic treatment), alleviating the disease, disorder, or condition, reducing the severity of the disease, disorder, or condition, stabilizing (i.e., not worsening) the disease, disorder, or condition, preventing the spread of the disease, disorder, or condition, delaying or slowing the progression of the disease, disorder, or condition, ameliorating or alleviating the disease, disorder, or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[0178] As used herein, the term "combination therapy" refers to a treatment regimen for a clinical indication that includes two or more therapeutic agents. Thus, the term refers to a treatment regimen in which a first therapy containing a first composition (e.g., active ingredient) is administered to a patient in conjunction with a second therapy containing a second composition (active ingredient) intended to treat the same or overlapping disease or clinical condition. Both the first and second compositions may act on the same or distinct cellular targets. The phrase "in conjunction with" in the context of combination therapy means that the therapeutic effect of the first therapy overlaps temporally and / or spatially with the therapeutic effect of the second therapy in a subject receiving the combination therapy. Thus, combination therapy can be formulated as a single formulation for simultaneous administration of the therapies or as separate formulations for sequential administration of the therapies.

[0179] As used herein, the term "alkyl" refers to a saturated monovalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Monovalent" means that the alkyl has one point of attachment to the rest of the molecule. In one embodiment, an alkyl has 1 to 12 carbon atoms (i.e., C 1-12 alkyl) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, alkyl has 1 to 8 carbon atoms (i.e., C 1-8alkyl), 1 to 7 carbon atoms (i.e., C 1-7 alkyl), 1 to 6 carbon atoms (i.e., C 1-6 alkyl), 1 to 4 carbon atoms (i.e., C 1-4 alkyl), or 1 to 3 carbon atoms (i.e., C 1-3 Examples include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. 1-6 Alkyl," "linear or branched C 1-4 Alkyl" or "Straight or branched C 1-3 Linear or branched alkyl, such as "alkyl," means that the saturated monovalent hydrocarbon radical is straight-chained or branched. As used herein, the term "straight-chained" in reference to an aliphatic hydrocarbon chain means that the chain is unbranched.

[0180] As used herein, the term "alkylene" refers to a saturated divalent hydrocarbon radical of 1 to 20 carbon atoms (i.e., C 1-20 "Alkylene" refers to an alkylene group, examples of which include, but are not limited to, those having the same core structure as the alkyl groups illustrated above. "Divalent" means that the alkylene has two points of attachment to the rest of the molecule. In one embodiment, alkylene is an alkylene group having 1 to 12 carbon atoms (i.e., C 1-12 alkylene) or 1 to 10 carbon atoms (i.e., C 1-10 In one embodiment, the alkylene has 1 to 8 carbon atoms (i.e., C 1-8 alkylene), 1 to 7 carbon atoms (i.e., C1-7 alkylene), 1 to 6 carbon atoms (i.e., C 1-6 alkylene), 1 to 4 carbon atoms (i.e., C 1-4 alkylene), 1 to 3 carbon atoms (i.e., C 1-3 alkylene), ethylene, or methylene. 1-6 alkylene," "linear or branched C 1-4 Alkylene" or "linear or branched C 1-3 Linear or branched alkylene, such as "alkylene," means that the saturated divalent hydrocarbon radical is linear or branched.

[0181] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical having one or more (e.g., 1 or 2) carbon-carbon double bonds, and alkenyl radicals include radicals having "cis" and "trans" configurations, or, alternatively, "E" and "Z" configurations.

[0182] As used herein, "alkenylene" refers to an aliphatic divalent hydrocarbon radical of 2 to 20 carbon atoms having one or two carbon-carbon double bonds (i.e., C 2-20 "Alkenylene" refers to an alkylene group having 2 to 12 carbon atoms (i.e., C 2-16 alkenylene), 2 to 10 carbon atoms (i.e., C 2-10 In one embodiment, the alkenylene has 2 to 4 carbon atoms (C 2-4 Examples include, but are not limited to, ethylene or vinylene (-CH=CH-), allyl (-CHCH=CH-), etc. 2-6 Alkenylene," "linear or branched C 2-4 Alkenylene" or "linear or branched C 2-3Linear or branched alkenylene, such as "alkenylene," means that the unsaturated divalent hydrocarbon radical is linear or branched.

[0183] As used herein, "cycloalkylene" refers to a divalent saturated carbocyclic ring radical having 3 to 12 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. "Divalent" means that the cycloalkylene has two points of attachment to the rest of the molecule. In one embodiment, the cycloalkylene is a 3- to 7-membered monocyclic or a 3- to 6-membered monocyclic ring. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, cyclononylene, cyclodecylene, cycloundecylene, cyclododecylene, and the like. In one embodiment, the cycloalkylene is cyclopropylene.

[0184] The terms "heterocycle," "heterocyclyl," heterocyclic, and "heterocyclic ring" are used interchangeably herein and refer to a cyclic group containing at least one N atom, a heteroatom selected from N and S, and optionally 1 to 3 additional heteroatoms, that is non-aromatic (i.e., partially saturated or fully saturated). It can be monocyclic or bicyclic (bridged or fused). Examples of heterocyclic rings include, but are not limited to, aziridinyl, diaziridinyl, thiaaziridinyl, azetidinyl, diazetidinyl, triazetidinyl, thiadiazetidinyl, thiazetidinyl, pyrrolidinyl, pyrazolidinyl, imidazolinyl, isothiazolidinyl, thiazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, azepanyl, azocanyl, and the like. A heterocycle contains 1 to 4 heteroatoms, which may be the same or different, selected from N and S. In one embodiment, the heterocycle contains 1 to 3 N atoms. In another embodiment, the heterocycle contains 1 or 2 N atoms. In another embodiment, the heterocycle contains 1 N atom. "4- to 8-membered heterocyclyl" refers to a radical having 4 to 8 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. "5- or 6-membered heterocyclyl" refers to a radical having 5 or 6 atoms (including 1 to 4 heteroatoms selected from N and S, or 1 to 3 N atoms, or 1 or 2 N atoms, or 1 N atom) arranged in a monocyclic ring. The term "heterocycle" is intended to include all possible isomeric forms.Heterocycles are described in Paquette, Leo A., Principles of Modern Heterocyclic Chemistry (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; The Chemistry of Heterocyclic Compounds, A Series of Monographs (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. Heterocyclyl groups may be carbon (carbon-linked) or nitrogen (nitrogen-linked) attached to the remainder of the molecule, where such is possible.

[0185] When a group is described as "optionally substituted," the group can be either (1) unsubstituted or (2) substituted. When a carbon of a group is described as optionally substituted with one or more of a list of substituents, one or more of the hydrogen atoms on the carbon (to the extent present) can be replaced separately and / or together with any independently selected substituents.

[0186] Suitable substituents for alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are those that do not significantly adversely affect the biological activity of the molecule. Unless otherwise specified, exemplary substituents for these groups include linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms; aryl; heteroaryl; heterocyclyl; halogen; guanidinium [—NH(C═NH)NH]; —OR 100 ;NR 101 R 102 ;-NO2;--NR 101 COR 102 ;-SR 100 ;--SOR 101 Sulfoxides represented by -SO2R 101 Sulfonic acid - SO3M; sulfuric acid - OSO3M; --SO2NR 101 R102 Sulfonamide; cyano; azide; -COR 101 ;-OCOR 101 ;--OCONR 101 R 102 and polyethylene glycol units (-OCH2CH2) n R 101 wherein M is H or a cation (Na + or K + ) etc.), and R 101 , R 102 , and R 103 are each independently H- a linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms; a polyethylene glycol unit (-OCH2CH2) n -R 104 wherein n is an integer from 1 to 24; an aryl having 6 to 10 carbon atoms; a heterocyclic ring having 3 to 10 carbon atoms; and a heteroaryl having 5 to 10 carbon atoms; and R 104 is H or a linear or branched alkyl having 1 to 4 carbon atoms, and R 100 , R 101 , R 102 , R 103 , and R 104 The alkyl, alkenyl, alkynyl, aryl, heteroaryl, and heterocyclyl in the group represented by the formula (I) are optionally substituted with one or more (e.g., 2, 3, 4, 5, 6, or more) substituents independently selected from halogen, —OH, —CN, —NO2, and unsubstituted linear or branched alkyl having 1 to 4 carbon atoms. Preferably, the substituents of the above optionally substituted alkyl, alkylene, alkenylene, cycloalkylene, and heterocyclyl are halogen, —CN, —NR 101 R 102 , -CF3, -OR 100 , aryl, heteroaryl, heterocyclyl, -SR 101 , -SOR 101 , -SO2R 101 Alternatively, suitable substituents are selected from the group consisting of halogen, —OH, —NO, —CN, C1-4 Alkyl, -OR 100 , N.R. 101 R 102 , --NR 101 COR 102 , -SR 100 , -SO2R 101 , -SO2NR 101 R 102 , --COR 101 , -OCOR 101 , and -OCONR 101 R 102 wherein R 100 , R 101 , and R 102 are each independently -H or C 1-4 It is alkyl.

[0187] As used herein, "halogen" refers to F, Cl, Br, or I. "Cyano" is --CN.

[0188] "Amine" or "amino," as used interchangeably herein, refers to a functional group containing a basic nitrogen atom bearing a lone pair of electrons.

[0189] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ionizable lipid of the present disclosure. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, acid tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. In cases where multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0190] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, processes, and their respective components that are essential to the process, method, or composition, but which embrace the inclusion of unspecified elements, whether essential or not. The use of "comprising" indicates inclusion rather than limitation.

[0191] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any element not recited in the description of the embodiment.

[0192] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic novel or functional characteristics of that embodiment of the present disclosure.

[0193] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. 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.

[0194] The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0195] Except in the examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are understood to be modified in all instances by the term "about." When used in connection with percentages, the term "about" can mean ±1%. The present disclosure is further illustrated by the following examples, but the scope of the present disclosure should not be limited thereto.

[0196] Grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. For reasons of convenience and / or patentability, one or more members of a group may be included in, or deleted from, a group. When any such inclusion or deletion occurs, the specification herein is deemed to include the group as modified and, therefore, to satisfy the specification of all Markush groups used in the appended claims.

[0197] In some embodiments of any of its aspects, the disclosure described herein does not relate to processes for cloning humans, processes for modifying the germline genetic identity of humans, the use of human embryos for industrial or commercial purposes, or processes for modifying the genetic identity of animals that are likely to cause suffering to humans or animals without providing any substantial medical benefit to them, and similarly processes for modifying the genetic identity of animals resulting from such processes.

[0198] Other terms are defined herein within the description of various aspects of the disclosure.

[0199] All patents and other publications, including literature references, issued patents, published patent applications, and copending patent applications, cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications, which might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute an admission as to the correctness of the dates or contents of these documents.

[0200] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While certain specific embodiments and examples of the present disclosure are described herein for illustrative purposes, those skilled in the art will recognize that various equivalent modifications are possible within the scope of the present disclosure. For example, while method steps or functions are presented in a given order, alternative embodiments may perform the functions in a different order, or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein can be applied to other procedures or methods, where appropriate. The various embodiments described herein can be combined to provide further embodiments. Where necessary, aspects of the present disclosure can be modified to employ compositions, functions, and concepts from the above references and applications to provide still further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting biological or chemical activity in terms of type or amount. These and other modifications can be made to the present disclosure in light of the Detailed Description. All such modifications are intended to be within the scope of the appended claims.

[0201] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements of other embodiments. Furthermore, although advantages associated with certain embodiments of the present disclosure are described in connection with those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages to fall within the scope of the present disclosure.

[0202] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting in any way. It is understood that the disclosure is not limited in any way to the particular methodology, protocols, and reagents, etc., described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the disclosure, which is defined solely by the claims.

[0203] II. Lipid Nanoparticles (LNPs) Provided herein are lipid nanoparticles (LNPs) comprising a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, and a helper lipid (e.g., DSPC). Also provided herein are LNPs consisting essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer, and a helper lipid (e.g., DSPC). Also provided herein are LNPs consisting of a therapeutic nucleic acid (TNA), an ionizable lipid, a structured lipid (e.g., a sterol), and one or more lipid-anchored polymers, e.g., a first lipid-anchored polymer and a second lipid-anchored polymer (e.g., DSPC). Also provided herein are LNPs comprising a therapeutic nucleic acid (TNA), an ionizable lipid, a structural lipid (e.g., a sterol), and one or more lipid-anchored polymers (e.g., a first lipid-anchored polymer and a second lipid-anchored polymer), but no helper lipid (e.g., 0 mol% helper lipid, such as DSPC). In some embodiments, the LNPs of the present disclosure do not comprise distearoylphosphatidylcholine (DSPC), or a salt or ester thereof, or a deuterated analog of any of the foregoing is present.

[0204] A. Ionizable lipids In some embodiments, the ionizable lipids are present in an LNP provided by the present disclosure at about 20 mol% to about 70 mol%, about 20 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 20 mol% to about 55 mol%, about 20 mol% to about 50 mol%, about 25 mol% to about 70 mol%, about 25 mol% to about 65 mol%, about 25 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 25 mol% to about 50 mol%, about 30 mol% to about 70 mol% of the total lipids present in the LNP. %, about 30 mol% to about 65 mol%, about 30 mol% to about 60 mol%, about 30 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 70 mol%, about 35 mol% to about 65 mol%, about 35 mol% to about 60 mol%, about 35 mol% to about 55 mol%, about 35 mol% to about 50 mol%, 40 mol% to about 70 mol%, about 40 mol% to about 65 mol%, about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, or about 40 mol% to about 50 mol%.

[0205] In some embodiments, the LNPs provided by the present disclosure comprise an ionizable lipid. Exemplary ionizable lipids in the LNPs of the present disclosure are those disclosed in International Patent Application Publication Nos. 2015 / 095340, 2015 / 199952, 2018 / 011633, 2017 / 049245, 2015 / 061467, 2012 / 040184, 2012 / 000104, 2015 / 074085, 2016 / 081029, 2017 / 004143, 2017 / 075531, 2017 / 117528, 2011 / 022460, and 2013 / 148 No. 541, No. 2013 / 116126, No. 2011 / 153120, No. 2012 / 044638, No. 2012 / 05 No. 4365, No. 2011 / 090965, No. 2013 / 016058, No. 2012 / 162210, No. 2008 / 0 No. 42973, No. 2010 / 129709, No. 2010 / 144740, No. 2012 / 099755, No. 2013 / No. 049328, No. 2013 / 086322, No. 2013 / 086373, No. 2011 / 071860, No. 2009 / 132131, 2010 / 048536, 2010 / 088537, 2010 / 054401, 201 No. 0 / 054406, No. 2010 / 054405, No. 2010 / 054384, No. 2012 / 016184, No. 20 09 / 086558, 2010 / 042877, 2011 / 000106, 2011 / 000107, 2 005 / 120152, 2011 / 141705, 2013 / 126803, 2006 / 007712, Nos. 2011 / 038160, 2005 / 121348, 2011 / 066651, 2009 / 127060, 2011 / 141704, 2006 / 069782, 2012 / 031043, 2013 / 006825, 2013 / 033563, 2013 / 089151, 2017 / 099823, 2015 / 095346, and 2013 / 086354, and U.S. Patent Application Publication Nos. 2016 / 0311759 and 2015 / 0376115,Same No. 2016 / 0151284, No. 2017 / 0210697, No. 2015 / 0140070, No. 2013 / 0178541, No. 2013 / 0303587, No. 2015 / 0141678, No. 201 5 / 0239926, 2016 / 0376224, 2017 / 0119904, 2012 / 0149894, 2015 / 0057373, 2013 / 0090372, 2013 / 0274 No. 523, No. 2013 / 0274504, No. 2013 / 0274504, No. 2009 / 0023673, No. 2012 / 0128760, No. 2010 / 0324120, No. 2014 / 0200257, Same No. 2015 / 0203446, No. 2018 / 0005363, No. 2014 / 0308304, No. 2013 / 0338210, No. 2012 / 0101148, No. 2012 / 0027796, No. 2012 / 0058144, 2013 / 0323269, 2011 / 0117125, 2011 / 0256175, 2012 / 0202871, 2011 / 0076335, 2006 / 0083 780, 2013 / 0123338, 2015 / 0064242, 2006 / 0051405, 2013 / 0065939, 2006 / 0008910, 2003 / 0022649, Nos. 2010 / 0130588, 2013 / 0116307, 2010 / 0062967, 2013 / 0202684, 2014 / 0141070, 2014 / 0255472, 2014 / 0039032, 2018 / 0028664, 2016 / 0317458, and 2013 / 0195920, the contents of all of which are incorporated herein by reference in their entireties.

[0206] Formula (A) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (A): [ka] (In the formula, R1 and R 1’ are each independently, C 1-3 is alkylene, R 2 and R 2’ are each independently a linear or branched C 1-6 Alkylene, or C 3-6 is cycloalkylene, R 3 and R 3’ each independently represents an optionally substituted C 1-6 Alkyl or optionally substituted C 3-6 is cycloalkyl, or Alternatively, R 2 Branched C 1-6 alkylene, and R 3 C 1-6 If it is alkyl, R 2 and R 3 together with the intervening N atom to form a 4- to 8-membered heterocyclyl, or Alternatively, R 2’ Branched C 1-6 alkylene, and R 3’ C 1-6 If it is alkyl, R 2’ and R 3’ together with the intervening N atom to form a 4- to 8-membered heterocyclyl; R 4 and R 4’ are each independently -CH, -CHCH, or -(CH)CH; R 5 and R 5’ are each independently hydrogen, C 1-20 Alkylene or C 2-20 is alkenylene, R 6 and R 6’ But for each occurrence, independently, C 1-20 Alkylene, C 3-20 Cycloalkylene or C 2-20 is alkenylene, m and n are each independently an integer selected from 1, 2, 3, 4, and 5, or a pharmaceutically acceptable salt thereof.

[0207] In some embodiments, R 2 and R 2’ are each independently, C 1-3 It is alkylene.

[0208] In some embodiments, R 1 or R 1’ Linear or branched C represented by 1-3 Alkylene, R 2 or R 2’ Linear or branched C represented by 1-6 Alkylene, and optionally substituted linear or branched C 1-6 Each alkyl is optionally substituted with one or more halo and cyano groups.

[0209] In some embodiments, R 1 and R 2 Together, C 1-3 alkylene, and R 1’ and R 2’ Together, C 1-3 Alkylene, for example, ethylene.

[0210] In some embodiments, R 3 and R 3’ each independently represents an optionally substituted C 1-3 Alkyl, for example, methyl.

[0211] In some embodiments, R 4 and R 4’ are each -CH.

[0212] In some embodiments, R 2 is an optionally substituted branched C 1-6 alkylene, and R 2 and R 3taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 2’ is an optionally substituted branched C 1-6 alkylene, and R 2’ and R 3’ taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0213] In some embodiments, R 4 is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3 and R 4 taken together with their intervening N atom form a 5- or 6-membered heterocyclyl. In some embodiments, R 4’ is -C(R a )2CR a or -[C(R a )2]2CR a and R a is C 1-3 alkyl, and R 3’ and R 4’ taken together with their intervening N atom form a 5- or 6-membered heterocyclyl such as pyrrolidinyl or piperidinyl.

[0214] In some embodiments, R 5 and R 5’ are each independently, C 1-10 Alkylene or C 2-10 In one embodiment, R is alkenylene. 5 and R 5’ are each independently, C 1-8 Alkylene or C 1-6 It is alkylene.

[0215] In some embodiments, R 6 and R 6’ is independently generated for each occurrence of C1-10 Alkylene, C 3-10 Cycloalkylene, or C 2-10 In one embodiment, C is an alkenylene. 1-6 Alkylene, C 3-6 Cycloalkylene, or C 2-6 In one embodiment, C is an alkenylene. 3-10 Cycloalkylene or C 3-6 In some embodiments, m and n are each 3.

[0216] In some embodiments, the ionizable lipid in the LNPs of the present disclosure can be selected from any one of the lipids listed in Table 1 below, or a pharmaceutically acceptable salt thereof. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0217] Formula (B) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (B): [ka] (In the formula, a is an integer ranging from 1 to 20 (e.g., a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); b is an integer ranging from 2 to 10 (e.g., b is 2, 3, 4, 5, 6, 7, 8, 9, or 10); R 1 is absent or (C2-C 20 ) alkenyl, -C(O)O(C2-C 20 ) alkyl, and (C2-C 20 ) cyclopropyl substituted with alkyl; R 2 However, (C2-C 20 ) alkyl) or a pharmaceutically acceptable salt thereof.

[0218] In a second embodiment of formula (B), the ionizable lipid of formula (B) has the formula (B-1): [ka] wherein c and d are each independently an integer ranging from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and the remaining variables are as described in formula (B), or a pharmaceutically acceptable salt thereof.

[0219] In a third embodiment of Formula (B), c and d in Formula (B-1) are each independently an integer ranging from 2 to 8, from 3 to 8, from 3 to 7, from 3 to 6, from 3 to 5, from 4 to 8, from 4 to 7, from 4 to 6, from 5 to 8, from 5 to 7, or from 6 to 8, and the remaining variables are as described for Formula (B-1).

[0220] In a fourth embodiment of Formula (B), c in Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B). Alternatively, c and d in Formula (B-1) are each independently 1, 3, 5, or 7, and the remaining variables are as described for Formula (B), or the second or third embodiment of Formula (B).

[0221] In a fifth embodiment of Formula (B), d in the cationic lipid of Formula (B-1) is 2, 3, 4, 5, 6, 7, or 8, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B). Alternatively, at least one of c and d in Formula (B-1) is 7, with the remaining variables being as described for Formula (B), or the second, third, or fourth embodiment of Formula (B).

[0222] In a sixth embodiment of formula (B), the ionizable lipid of formula (B) or formula (B-1) is of formula (B-2): [ka] wherein the remaining variables are as described for Formula (B) or Formula (B-1), or a pharmaceutically acceptable salt thereof.

[0223] In a seventh embodiment of Formula (B), b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 9, 4 to 8, 4 to 7, 4 to 6, 5 to 9, 5 to 8, 5 to 7, 6 to 9, 6 to 8, or 7 to 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B). Alternatively, b in Formula (B), Formula (B-1), or Formula (B-2) is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, or sixth embodiment of Formula (B).

[0224] In an eighth embodiment of Formula (B), a in Formula (B), Formula (B-1), or Formula (B-2) is an integer ranging from 2 to 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in formula (B), formula (B-1), or formula (B-2) is 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 4 to 18, 4 to 17, 4 to 16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 25-8, 5-7, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 7-18, 7-17, 7-16, 7 ~15, 7~14, 7~13, 7~12, 7~11, 7~10, 7~9, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 11~18, 11~17, 11~16, 11~1 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 124, 125, 136, 136, 137, 146, 147, 148, 1518, 1517, or 1618, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B). Alternatively, a in Formula (B), Formula (B-1), or Formula (B-2) is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, or seventh embodiment of Formula (B).

[0225] In a ninth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 15 ) alkenyl, -C(O)O(C4-C 16 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). Alternatively, R in Formula (B), Formula (B-1), or Formula (B-2) is selected from 1 is absent or (C5-C 12 ) alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12 ) cyclopropyl substituted with alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). In another alternative, R in the cationic lipid of Formula (B), Formula (B-1), or Formula (B-2) is selected from: 1 is absent or (C5-C 10 ) alkenyl, -C(O)O(C4-C 10 ) alkyl, and (C4-C 10 ) cyclopropyl substituted with alkyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0226] In a tenth embodiment of formula (B), R 1 is C 10alkenyl, and the remainder of the variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B).

[0227] In an eleventh embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1 C(O)O(C2-C 20 ) alkyl, -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 ) alkyl is an unbranched alkyl, and the remaining variables are as described for Formula (B), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is —C(O)O(C alkyl). Alternatively, R in formula (B), formula (B-1), or formula (B-2) 1 -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 The alkyl in alkyl is a branched alkyl, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B). In one embodiment, R 1 is -C(O)O(C 17 alkyl), and the remainder of the variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, or ninth embodiment of Formula (B).

[0228] In a twelfth embodiment of formula (B), R in formula (B), formula (B-1), or formula (B-2) 1is selected from any group listed in Table 2 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). The present disclosure provides a method for treating R 1 and any one of the groups R in Table 3 of formula (B) 2 Further contemplated are combinations with any one of the groups, wherein the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, or eighth embodiment of Formula (B). [Table 2]

[0229] In a thirteenth embodiment, R in formula (B) 2 or a pharmaceutically acceptable salt thereof, selected from any of the groups listed in Table 3 below, where the wavy bond in each group indicates the point of attachment of the group to the remainder of the ionizable lipid molecule, and the remaining variables are as described for Formula (B), Formula (B-1), or Formula (B-2), or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth embodiment of Formula (B). [Table 3]

[0230] Table 4 below provides specific examples of ionizable lipids of Formula (B), including pharmaceutically acceptable salts, as well as ionized and neutral forms. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5]

[0231] Formula (C) In some embodiments, the ionizable lipid in the LNPs of the present disclosure has formula (C): [ka] (In the formula, R 1 and R 1’ are each independently R a and (C1-C6) alkylene optionally substituted with one or more groups selected from R 2 and R 2’ are each independently (C1-C2) alkylene; R 3 and R 3’ are each independently R b or (C1-C6) alkyl optionally substituted with one or more groups selected from Alternatively, R 2 and R 3 and / or R 2’ and R 3’ together with the intervening N atom to form a 4- to 7-membered heterocyclyl; R 4 and R 4 each ' is (C2-C6)alkylene interrupted by -C(O)O-; R 5 and R 5 each independently being optionally interrupted by —C(O)O— or (C3-C6)cycloalkyl, (C2-C 30 ) alkyl or (C2-C 30 ) alkenyl, R a and R b wherein each is halo or cyano, or a pharmaceutically acceptable salt thereof.

[0232] In a second embodiment of formula (C), R 1 and R 1 are each independently (C-C) alkylene, and the remaining variables are as described above for formula (C). Alternatively, R 1 and R 1’ is independently at each occurrence (C1-C3) alkylene, and the remainder of the variables are as described above for formula (C).

[0233] In a third embodiment of formula (C), the ionizable lipid of formula (C) has the formula (C-1): [ka] (In the formula, R 2 and R 2’ , R 3 and R 3’ , R 4 and R 4 ', as well as R 5 and R 5 ' is represented by Formula (C), or as described above for the second embodiment of Formula (C), or a pharmaceutically acceptable salt thereof.

[0234] In a fourth embodiment, the ionizable lipid of formula (C) has formula (C-2) or formula (C-3): [ka] (In the formula, R 4 and R 4 ' and R 5 and R 5 ' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0235] In a fifth embodiment of formula (C), the ionizable lipid of formula (C) is of formula (C-4) or formula (C-5): [ka] (In the formula, R 5and R 5 ' is as described above for formula (C)), or a pharmaceutically acceptable salt thereof.

[0236] In a sixth embodiment of Formula (C), the ionizable lipid of Formula (C) is Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9): [ka] (In the formula, R 5 and R 5 ' is as described above for formula (XV)), or a pharmaceutically acceptable salt thereof.

[0237] In a seventh embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 and R 5’ is a branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is branched alkyl or branched alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’is branched alkyl or branched alkenyl, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0238] In an eighth embodiment of formula (C), R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C-C)cycloalkyl, (C-C 26 ) alkyl or (C6-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C6-C 26 ) alkyl or (C6-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C7-C 26 ) alkyl or (C7-C 26 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or (C3-C5)cycloalkyl, (C8-C 26 ) alkyl or (C8-C 26) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 are each optionally interrupted by —C(O)O— or cyclopropyl, (C-C 24 ) alkyl or (C6-C 24 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 24 ) alkyl or (C8-C 24 ) alkenyl, and the (C8-C 24 )alkyl is optionally interrupted by -C(O)O- or cyclopropyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C8-C 10 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by cyclopropyl (C 14 -C 16 ) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is interrupted by -C(O)O- (C 10 -C 24) alkyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 is (C 16 -C 18 ) alkenyl, and the remaining variables are as described above for Formula (C), or the second embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5 -(CH2)3C(O)O(CH2)8CH3, -(CH2)5C(O)O(CH2)8CH3, -(CH2)7C(O)O(CH2)8CH3, - (CH2)7C(O)OCH[(CH2)7CH3]2, -(CH2)7-C3H6-(CH2)7CH3, -(CH2)7CH3, -(CH2)9CH 3、 -(CH2) 16 CH3, -(CH2)7CH=CH(CH2)7CH3, or -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and the remainder of the variables are as described above for Formula (C), or the second embodiment of Formula (C).

[0239] In the ninth embodiment, R in formula (C), formula (C-1), formula (C-2), formula (C-3), formula (C-4), formula (C-5), formula (C-6), formula (C-7), formula (C-8), or formula (C-9) 5’ is interrupted by -C(O)O- (C 15 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 28) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 28 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 17 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 19 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R in Formula (C), Formula (C-1), Formula (C-2), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-6), Formula (C-7), Formula (C-8), or Formula (C-9) 5’ is interrupted by -C(O)O- (C 20 -C 26 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’ is interrupted by -C(O)O- (C 22 -C 24 ) alkyl, and the remaining variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C). Alternatively, R 5’is —(CH2)5C(O)OCH[(CH2)7CH3]2, —(CH2)7C(O)OCH[(CH2)7CH3]2, —(CH2)5C(O)OCH(CH2)2[(CH2)7CH3]2, or —(CH2)7C(O)OCH(CH2)2[(CH2)7CH3]2, and the remainder of the variables are as described above for Formula (C), or the second or eighth embodiment of Formula (C).

[0240] In some embodiments, the ionizable lipid of Formula (C), Formula (C-1), Formula (C-3), Formula (C-3), Formula (C-4), Formula (C-5), Formula (C-7), Formula (C-8), or Formula (C-9) may be selected from any of the lipids listed in Table 5 below, or a pharmaceutically acceptable salt thereof. [Table 5-1] [Table 5-2]

[0241] Formula (D) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has formula (D): [ka] (In the formula, R' is absent, hydrogen, or C1-C6 alkyl, provided that when R' is hydrogen or C1-C6 alkyl, R', R 1 , and R 2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen, C1-C6 alkyl, or C2-C6 alkenyl; R 3 But C1-C 12 Alkylene or C2-C 12 is alkenylene, R 4 But C1-C 18 Unbranched alkyl, C2-C18 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C8 alkylene, or C2-C8 alkenylene; R 6a and R 6b are each independently, C7-C 16 Alkyl or C7-C 16 alkenyl, provided that R 6a and R 6b provided that the total number of carbon atoms in X 1 and X 2 each independently represents -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0242] In a second embodiment of formula (D), X 1 and X 2 is the same, and all other remaining variables are as described for formula (C).

[0243] In a third embodiment of formula (D), X 1 and X 2 are each independently -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-; or X 1 and X 2 are each independently -C(=O)O-, -C(=O)S-, or -SS-, or X 1 and X 2 is each independently -C(=O)O- or -SS-, and all other remaining variables are as described for Formula (D), or the second embodiment of Formula (D).

[0244] In a fourth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (D-1): [ka] wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0245] In a fifth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-2): [ka] wherein n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0246] In a sixth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-3): [ka] wherein all other remaining variables are as described for Formula (D), or the second or third embodiment of Formula (D), or a pharmaceutically acceptable salt thereof.

[0247] In a seventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D), R 1 and R 2 are each independently hydrogen, C1-C6 alkyl or C2-C6 alkenyl, or C1-C5 alkyl or C2-C5 alkenyl, or C1-C4 alkyl or C2-C4 alkenyl, or C6 alkyl, or C5 alkyl, or C4 alkyl, or C3 alkyl, or C2 alkyl, or C1 alkyl, or C6 alkenyl, or C5 alkenyl, or C4 alkenyl, or C3 alkenyl, or C2 alkenyl, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or the second or third embodiment of Formula (D).

[0248] In an eighth embodiment of Formula (D), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (D-4): [ka] wherein all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D), or a pharmaceutically acceptable salt thereof.

[0249] In a ninth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 3 is C1-C9 alkylene or C2-C9 alkenylene, C1-C7 alkylene or C2-C7 alkenylene, C1-C5 alkylene or C2-C5 alkenylene, or C2-C8 alkylene or C2-C8 alkenylene, or C3-C7 alkylene or C3-C7 alkenylene, or C5-C7 alkylene or C5-C7 alkenylene, or R 3 is C 12 Alkylene, C 11 Alkylene, C 10 Alkylene, C9 alkylene, or C8 alkylene, or C7 alkylene, or C6 alkylene, or C5 alkylene, or C4 alkylene, or C3 alkylene, or C2 alkylene, or C1 alkylene, or C 12 Alkenylene, C 11 Alkenylene, C 10 alkenylene, C9 alkenylene, or C8 alkenylene, or C7 alkenylene, or C6 alkenylene, or C5 alkenylene, or C4 alkenylene, or C3 alkenylene, or C2 alkenylene, and all other remaining variables are as described for the second, third, or seventh embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0250] In a tenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, or seventh embodiment of Formula (D), R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, or ninth embodiment of Formula (D).

[0251] In an eleventh embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, or tenth embodiment of Formula (D), R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is a C5-C7 unbranched alkyl or a C5-C7 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, third, seventh, ninth, or tenth embodiment of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or Formula (D).

[0252] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D), R 6a and R 6b are each independently C6-C 14 Alkyl or C6-C 14 alkenyl, or R 6a and R 6b are each independently C8-C 12 Alkyl or C8-C 12 alkenyl, or R 6a and R 6b are each independently, C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, or eleventh embodiment of Formula (D).

[0253] In a thirteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D), or a pharmaceutically acceptable salt thereof, R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 16 Alkyl, C 15 Alkyl, C 14 Alkyl, C 13 Alkyl, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 16 Alkenyl, C 15 Alkenyl, C 14 Alkenyl, C 13 Alkenyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, provided that R 6a and R 6b is greater than 15, and all other remaining variables are as described for Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (D).

[0254] In a fourteenth embodiment of Formula (D), in an ionizable lipid, e.g., a cationic lipid, according to Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), Formula (D-4), or the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (D), R as defined in any one of the preceding embodiments. 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl or R 6a is C8 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 11 alkyl or R 6a is a C7 alkyl, and R6a is a C9 alkyl or R 6a is a C9 alkyl, and R 6a is a C7 alkyl or R 6a is C8 alkyl, and R 6a C 10 alkyl or R 6a C 10 alkyl, and R 6a is C8 alkyl or R 6a is a C9 alkyl, and R 6a C 11 alkyl or R 6a C 11 alkyl, and R 6a is a C9 alkyl or R 6a C 10 alkyl, and R 6a C 12 alkyl or R 6a C 12 alkyl, and R 6a C 10 alkyl or R 6a C 11 alkyl, and R 6a C 13 alkyl or R 6a C 13 alkyl, and R 6a C 11 is alkyl, and all other remaining variables are as described for the second, third, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula I, Formula II, Formula III, Formula IV, Formula V, or Formula (D).

[0255] In a fifteenth embodiment of formula (D), R 4 is C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4bis as described above for the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth embodiment of Formula (D).

[0256] In one embodiment, the ionizable lipid of the present disclosure, e.g., a cationic lipid, or an ionizable lipid of Formula (D), Formula (D-1), Formula (D-2), Formula (D-3), or Formula (D-4), is any one lipid selected from the lipids listed in Table 6 below, or a pharmaceutically acceptable salt thereof. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0257] In one embodiment, the ionizable lipid in the LNPs of the disclosure is lipid number 87: [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0258] Formula (E) In some embodiments, the ionizable lipid, e.g., cationic lipid, in the LNPs of the present disclosure has formula (D): [ka] (In the formula, R' is absent, hydrogen, or C1-C3 alkyl, provided that when R' is hydrogen or C1-C3 alkyl, R', R 1 , and R2 provided that all nitrogen atoms to which they are attached are positively charged, R 1 and R 2 are each independently hydrogen or C1-C3 alkyl; R 3 But C3-C 10 Alkylene or C3-C 10 is alkenylene, R 4 But C1-C 16 Unbranched alkyl, C2-C 16 unbranched alkenyl, or [ka] where: R 4a and R 4b are each independently, C1-C 16 Unbranched alkyl or C2-C 16 is an unbranched alkenyl, R 5 is absent, C1-C6 alkylene, or C2-C6 alkenylene; R 6a and R 6b are each independently, C7-C 14 Alkyl or C7-C 14 is alkenyl, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, -SS-, -C(R a )=N-, -N=C(R a )-, -C(R a )=NO-, -ON=C(R a )-, -C(=O)NR a -, -NR a C(=O)-, -NR a C(=O)NR a -, -OC(=O)O-, -OSi(R a )2O-, -C(=O)(CR a 2) C(=O)O- or OC(=O)(CR a 2) C(=O)-, wherein: R a is independently at each occurrence hydrogen or C1-C6 alkyl; and n is an integer selected from 1, 2, 3, 4, 5, and 6, or a pharmaceutically acceptable salt thereof.

[0259] In a second embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid according to the first embodiment, or a pharmaceutically acceptable salt thereof, X is -OC(=O)-, -SC(=O)-, -OC(=S)-, -C(=O)O-, -C(=O)S-, or -SS-, and all other remaining variables are as described for Formula I or the first embodiment.

[0260] In a third embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-1): [ka] wherein n is an integer selected from 1, 2, 3, and 4, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof. Alternatively, n is an integer selected from 1, 2, and 3, and all other remaining variables are as described for Formula (E), or the second embodiment of Formula (E).

[0261] In a fourth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-2): [ka] wherein all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E), or a pharmaceutically acceptable salt thereof.

[0262] In a fifth embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, in an LNP of the present disclosure, R1 and R 2 are each independently hydrogen or C1-C2 alkyl or C2-C3 alkenyl, or R', R 1 , and R 2 are each independently hydrogen, C1-C2 alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), or the second embodiment of Formula (E).

[0263] In a sixth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure is represented by Formula (E-3): [ka] wherein all other remaining variables are as described for the second or fifth embodiment of Formula (E), Formula (E-1), Formula (E-2), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0264] In a seventh embodiment of Formula (E), in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E), R 5 is absent or C1-C8 alkylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent, C-C alkylene, or C-C alkenylene, or R 5 is absent or R 5 is C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, C alkenylene, or C alkenylene, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), or the second or fifth embodiment of Formula (E).

[0265] In an eighth embodiment of Formula (E), the ionizable lipid, e.g., cationic lipid, in the LNP of the present disclosure has the formula (E-4): [ka] wherein all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), (E-3), or Formula (E), or a pharmaceutically acceptable salt thereof.

[0266] In a ninth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, or seventh embodiment of Formula (E), or a pharmaceutically acceptable salt thereof, R 4 is C1-C 14 Unbranched alkyl, C2-C 14 unbranched alkenyl, or [ka] (In the formula, R 4a and R 4b are each independently, C1-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl), or R 4 is C2-C 12 Unbranched alkyl or C2-C 12 unbranched alkenyl, or R 4 is C5-C 12 Unbranched alkyl or C5-C 12 unbranched alkenyl, or R 4 is C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 unbranched alkyl, C1 unbranched alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C 14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl, or R 4 teeth, [ka] (R in the formula 4a and R 4b are each independently, C2-C 10 Unbranched alkyl or C2-C 10 unbranched alkenyl), or R 4 teeth, [ka] (In the formula, R 4a and R 4b are each independently, C 16 Unbranched alkyl, C 15 Unbranched alkyl, C 14 Unbranched alkyl, C 13 Unbranched alkyl, C 12 Unbranched alkyl, C 11 Unbranched alkyl, C 10 Unbranched alkyl, C9 unbranched alkyl, C8 unbranched alkyl, C7 unbranched alkyl, C6 unbranched alkyl, C5 unbranched alkyl, C4 unbranched alkyl, C3 unbranched alkyl, C2 alkyl, C1 alkyl, C 16 Unbranched alkenyl, C 15 Unbranched alkenyl, C14 Unbranched alkenyl, C 13 Unbranched alkenyl, C 12 Unbranched alkenyl, C 11 Unbranched alkenyl, C 10 unbranched alkenyl, C9 unbranched alkenyl, C8 unbranched alkenyl, C7 unbranched alkenyl, C6 unbranched alkenyl, C5 unbranched alkenyl, C4 unbranched alkenyl, C3 unbranched alkenyl, or C2 alkenyl), and all other remaining variables are as described for the second, fifth, or seventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0267] In a tenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, or ninth embodiment of Formula (E), R 3 is C3-C8 alkylene or alkenylene, C3-C7 alkylene or alkenylene, or C3-C5 alkylene or alkenylene, or R 3 is C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, or C alkenylene, and all other remaining variables are as described for the second, fifth, seventh, or ninth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0268] In an eleventh embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), R 6a and R 6b are each independently C7-C 12 Alkyl or C7-C12 alkenyl, or R 6a and R 6b are each independently C8-C 10 Alkyl or C8-C 10 alkenyl, or R 6a and R 6b are each independently, C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, or tenth embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0269] In a twelfth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), R 6a and R 6b contain an equal number of carbon atoms, or R 6a and R 6b are the same or R 6a and R 6b are both C 12 Alkyl, C 11 Alkyl, C 10 Alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C 12 Alkenyl, C 11 Alkenyl, C 10 alkenyl, C9 alkenyl, C8 alkenyl, or C7 alkenyl, and all other remaining variables are as described for the second, fifth, seventh, ninth, tenth, or eleventh embodiment of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or Formula (E).

[0270] In a thirteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), R as defined in any one of the preceding embodiments is 6a and R 6b each contain a different number of carbon atoms, or R 6a and R 6b The number of carbon atoms in R differs by one or two carbon atoms, or 6a and R 6b The number of carbon atoms in R differs by one carbon atom, or 6a is a C7 alkyl, and R 6a is C8 alkyl, R 6a is C8 alkyl, and R 6a is C7 alkyl, R 6a is C8 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6a is C8 alkyl, R 6a is a C9 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a C 10 R is alkyl 6a C 11 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 11 R is alkyl 6a is a C7 alkyl, and R 6a is C9 alkyl, R 6a is a C9 alkyl, and R 6a is C7 alkyl, R 6ais C8 alkyl, and R 6a C 10 R is alkyl 6a C 10 alkyl, and R 6a is C8 alkyl, R 6a is a C9 alkyl, and R 6a C 11 R is alkyl 6a C 11 alkyl, and R 6a is C9 alkyl, R 6a C 10 alkyl, and R 6a C 12 R is alkyl 6a C 12 alkyl, and R 6a C 10 is alkyl, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, or twelfth embodiment of Formula (E).

[0271] In a fourteenth embodiment, in an ionizable lipid, e.g., a cationic lipid, according to Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E), R' is absent, and all other remaining variables are as described for Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), or the second, fifth, seventh, ninth, tenth, eleventh, twelfth, or thirteenth embodiment of Formula (E).

[0272] In one embodiment, the ionizable lipid, e.g., cationic lipid, or cationic lipid of Formula (E), Formula (E-1), Formula (E-2), Formula (E-3), Formula (E-4), in the LNP of the present disclosure is any one lipid selected from the lipids in Table 7, or a pharmaceutically acceptable salt thereof. [Table 7-1] [Table 7-2] [Table 7-3]

[0273] Specific examples are provided in the Exemplification section below and are included as part of the cationic or ionizable lipids described herein. Pharmaceutically acceptable salts as well as neutral forms are also included.

[0274] Cleavable lipids In some embodiments, the LNPs provided by the present disclosure include an ionizable lipid that is also a cleavable lipid. As used herein, the term "cleavable lipid," which may be used interchangeably with the term "SS-cleavable lipid," refers to an ionizable lipid that contains a disulfide bond (SS). The SS in the cleavable lipid is the cleavable unit. In one embodiment, the cleavable lipid includes an amine, e.g., a tertiary amine, and, e.g., a disulfide bond. In this cleavable lipid, the amine can become protonated in an acidic compartment (e.g., an endosome or lysosome), resulting in LNP destabilization, and the cleavable lipid can become cleaved in a reducing environment (e.g., the cytoplasm). Cleavable lipids also include pH-activated lipid-like materials, such as ss-OP lipids, ssPalm lipids, ss-M lipids, ss-E lipids, ss-EC lipids, ss-LC lipids, and ss-OC lipids.

[0275] According to some embodiments, the SS-cleavable lipids are described in International Patent Application Publication No. WO 2019188867, which is incorporated herein by reference in its entirety.

[0276] In one embodiment, the cleavable lipid may comprise three components: an amine head group, a linker group, and a hydrophobic tail. In one embodiment, the cleavable lipid comprises one or more phenyl ester bonds, one or more tertiary amino groups, and a disulfide bond. The tertiary amine groups provide pH responsiveness and induce endosomal escape, the phenyl ester bonds enhance the degradability (autolysis) of the structure, and the disulfide bonds become cleaved in a reducing environment.

[0277] In one embodiment, the cleavable lipid is a ss-OP lipid. In one embodiment, the ss-OP lipid comprises the structure of lipid A shown below:

[0278] Lipid A [ka] In one embodiment, the SS-cleavable lipid is SS-cleavable pH-activated lipid-like material (ssPalm). ssPalm lipid is well known in the art. For example, see Togashi et al., Journal of Controlled Release, 279 (2018) 262-270, the entire contents of which are incorporated herein by reference. In one embodiment, the ssPalm is ssPalmM lipid, comprising the structure of lipid B shown below.

[0279] lipid B [ka] In one embodiment, the ssPalmE lipid is a ssPalmE-P4-C2 lipid comprising the structure of lipid C below:

[0280] lipid C [ka] In one embodiment, the ssPalmE lipid is a ssPalmE-Paz4-C2 lipid comprising the structure of lipid D below:

[0281] lipid D [ka] In one embodiment, the cleavable lipid is a ss-M lipid. In one embodiment, the ss-M lipid comprises the structure shown in lipid E ​​below.

[0282] Lipid E [ka] In one embodiment, the cleavable lipid is an ss-E lipid. In one embodiment, the ss-E lipid comprises the structure shown in lipid F below.

[0283] lipid F [ka] In one embodiment, the cleavable lipid is a ss-EC lipid. In one embodiment, the ss-EC lipid comprises the structure shown for lipid G below.

[0284] lipid G [ka] In one embodiment, the cleavable lipid is a ss-LC lipid. In one embodiment, the ss-LC lipid comprises the structure shown for lipid H below.

[0285] lipid H [ka] In one embodiment, the cleavable lipid is a ss-OC lipid. In one embodiment, the ss-OC lipid comprises the structure shown for lipid J below.

[0286] lipid J [ka]

[0287] Other lipids In some embodiments, the ionizable lipid in the LNPs of the present disclosure is N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DL EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylcarboxamidoethyl 1-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[N-( N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethylhydroxyethylammonium bromide (DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC).In some variations, the condensing agent, e.g., the cationic lipid, is, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleo ...-MC3-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-MC3-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA)], 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin- These lipids include diethylaminopropane (DODAP), 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA), morpholinocholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyldioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyldioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen).

[0288] In some embodiments, the ionizable lipid in the LNPs of the present disclosure has the following structure: [ka] [ka] [ka] or a pharmaceutically acceptable salt or ester thereof, or a deuterated analog thereof.

[0289] B. Structured lipids In some embodiments, LNPs provided by the present disclosure comprise structured lipids. Without wishing to be bound by any particular theory, it is believed that structured lipids, when present in LNPs, contribute to the membrane integrity and stability of the LNPs.

[0290] In some embodiments, the structured lipid is a sterol, such as cholesterol, or a derivative thereof. In one embodiment, the structured lipid is cholesterol. In another embodiment, the structured lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol, non-polar analogs such as 5α-cholestan, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate, and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments, the cholesterol derivative is cholestryl hemisuccinate (CHEMS).

[0291] Exemplary cholesterol derivatives are described in International Patent Application Publication No. 2009 / 127060 and US Patent Application Publication No. 2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0292] In some embodiments, the sterol in the LNPs of the present disclosure is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, and derivatives thereof, and any combination thereof. In one embodiment, the sterol is cholesterol. In another embodiment, the sterol is beta-sitosterol.

[0293] In some embodiments, structured lipids comprise about 20 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, structured lipids comprise about 25 mol% to about 45 mol% of the total lipid content of the LNP. In some embodiments, structured lipids comprise about 30 mol% to about 45% of the total lipids present in the LNP. In some embodiments, structured lipids comprise about 30 mol% to about 40 mol% of the total lipids present in the LNP. In some embodiments, such components are about 40 mol% of the total lipids present in the LNP. In some embodiments, structured lipids, e.g., sterols, comprise about 20 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, structured lipids, e.g., sterols, comprise about 30 mol% to about 40 mol% of the total lipids present in the LNP. In some embodiments, structured lipids, e.g., sterols, comprise about 35 mol% to about 40 mol% of the total lipids present in the LNP, and the average LNP size is about 60 nm to about 80 nm in diameter.

[0294] In some embodiments, the structured lipid is cholesterol and comprises about 30 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 35 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% to about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 45 mol% of the total lipids present in the LNP. In some embodiments, the structured lipid is cholesterol and comprises about 40 mol% to about 45 mol% of the total lipids present in the LNP, and the encapsulation efficiency ("Enc. Eff.") of the TNA is greater than 95%, and / or the average size of the LNP is in the range of about 70 nm to 90 nm in diameter.

[0295] C. Helper lipids In some embodiments, the LNPs provided by the present disclosure comprise a helper lipid.

[0296] In some embodiments, the helper lipid is DSPC, a salt or ester thereof, or a deuterated analog of any of the foregoing. In some embodiments, the helper lipid is DOPE, a salt or ester thereof, or a deuterated analog of any of the foregoing. In some embodiments, the helper lipid is ceramide, a salt or ester thereof, or a deuterated analog of any of the foregoing.

[0297] As used herein, the term "salt" refers to pharmaceutically acceptable salts of helper lipids, including both acid and base addition salts, which retain the biological effectiveness and properties of the free acid or base form of the helper lipid.

[0298] As used herein, the term "ester" refers to an ester of a helper lipid. As a non-limiting example, a hydroxyl group of a helper lipid can be linked to an organic acid such as a phosphoric acid or a carboxylic acid through an esterification process to form an ester (e.g., a carboxylate or a phosphate) of the helper lipid.

[0299] As used herein, "deuterated analog," when referring to a helper lipid, means an analog of a helper lipid in which any one or more hydrogen atoms of the helper lipid have been replaced with deuterium.

[0300] In some embodiments, the LNPs of the present disclosure do not contain or include a helper lipid (e.g., distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0301] In some embodiments, the helper lipid (e.g., a ceramide of the present disclosure) represents about 2 mol% to about 40 mol%, or about 5 mol% to about 40 mol%, or about 5 mol% to about 35 mol%, or about 5 mol% to about 30 mol%, or about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol%, or about 5 mol% to about 15 mol%, or 10 mol% to about 40 mol%, or about 10 mol% to about 35 mol%, or about 10 mol% to about 30 mol%, or about 10 mol% to about 25 mol%, or about 10 mol% to about 20 mol%, or 15 mol% to about 40 mol%, or about 15 mol% to about 35 mol%, or about 15 mol% to about 30 mol%, or about 15 mol% to about 25 mol%, or about 15 mol% to about 20 mol%, or 20 mol% to about 40 mol%, or about 20 mol% to about 35 mol%, or about 20 mol% to about 30 mol%, or about 20 mol% to about In some embodiments, the helper lipid (e.g., DSPC, DOPE, ceramide, etc.) comprises about 10 mol% to about 20 mol% of the total lipid present in the LNP, and such LNPs having about 10 mol% to about 20 mol% of the total lipid present in the LNP exhibit an overall increase in tolerability (e.g., as shown by a reduced weight loss profile and cytokine response in subjects) compared to LNPs containing less than 10% of the same helper lipid.

[0302] D. Lipid-anchored polymers In some embodiments, the LNPs provided by the present disclosure comprise at least one type of lipid-anchored polymer, e.g., a first lipid-anchored polymer. As used herein, the term "lipid-anchored polymer" refers to a molecule comprising a lipid moiety covalently attached to a polymer, e.g., via a linker. Without wishing to be bound by theory, it is believed that the lipid-anchored polymer can inhibit aggregation of the LNPs disclosed herein, provide steric stabilization of the LNPs in vivo, and increase their blood half-life (t1 / 2). In some embodiments, the LNPs provided by the present disclosure comprise two lipid-anchored polymers, i.e., a first lipid-anchored polymer and a second lipid-anchored polymer.

[0303] Lipid moieties in lipid-anchored polymers More specifically, in one embodiment, the lipid-anchored polymer, e.g., the first lipid-anchored polymer according to the present disclosure, comprises: (i) a polymer; (ii) a lipid moiety comprising at least one hydrophobic tail, which may be linear or branched; (iii) optionally, a linker connecting the polymer to the lipid moiety; At least one hydrophobic tail (which may be linear or branched) comprises 18 to 22 carbon atoms in the single aliphatic chain backbone, e.g., 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, a lipid-anchored polymer, e.g., a first lipid-anchored polymer, comprises a lipid moiety comprising a single or two hydrophobic tails, each of which comprises 18 to 22 carbon atoms in the single aliphatic chain backbone, e.g., 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone.

[0304] As used herein, the term "linker lipid moiety" refers to a lipid moiety comprising at least two hydrophobic tails, e.g., two hydrophobic tails, covalently attached to a linker. In some embodiments, the linker lipid moiety can be part of a lipid-anchored polymer.

[0305] In one embodiment, at least one (e.g., single or two) hydrophobic tail is a fatty acid (saturated or unsaturated). Non-limiting examples of at least one (e.g., single or two) hydrophobic tail containing 12 to 22 carbon atoms in a single aliphatic chain backbone include lauric acid, myristic acid, myristoleic acid, octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0306] The term "derivative," as used herein with respect to a hydrophobic tail in a lipid-anchored polymer, refers to a hydrophobic tail that has been modified compared to the original or native hydrophobic tail. In some embodiments, a derivative comprises one or more of the following modifications compared to the original or native hydrophobic tail: a) a carboxylate group has been replaced with an amine, amide, ether, or carbonate group; b) one or more saturation points, e.g., double bonds, have been introduced into the hydrophobic tail (e.g., via dehydrogenation); c) one or more saturation points, e.g., double bonds, have been removed from the hydrophobic tail (e.g., via hydrogenation); or d) if present, the configuration of one or more double bonds has been changed, e.g., from a cis configuration to a trans configuration or from a trans configuration to a cis configuration. A derivative comprises the same number of carbon atoms as its original or native hydrophobic tail.

[0307] As used herein, the term "single aliphatic chain backbone" refers to the main linear aliphatic chain or carbon chain, i.e., the longest continuous linear aliphatic chain or carbon chain, when referring to the hydrophobic tail in a lipid-anchored polymer. For example, the following alkyl chain with several branching points contains 18 carbon atoms in the single aliphatic chain backbone, i.e., the longest continuous linear alkyl chain contains 18 carbon atoms. Note that the one or two carbon atoms at the several branching points (all indicated by *) are not included in the number of carbon atoms in the single aliphatic chain backbone. [ka]

[0308] In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer according to the present disclosure comprises: (i) a polymer; (ii) a lipid moiety comprising at least two hydrophobic tails, which may be linear or branched; (iii) optionally, a linker connecting the polymer to the lipid moiety; At least two hydrophobic tails (which may be linear or branched) comprise 18 to 22 carbon atoms in the single aliphatic chain backbone, e.g., 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the lipid-anchored polymer or first lipid-anchored polymer comprises a lipid moiety comprising two hydrophobic tails, wherein the two hydrophobic tails each independently comprise 18 to 22 carbon atoms in the single aliphatic chain backbone, e.g., 18, 19, 20, 21, or 22 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently comprise 16 to 21 carbon atoms in the single aliphatic chain backbone, e.g., 16, 17, 18, 19, 20, or 21 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 20 carbon atoms in the single aliphatic chain backbone, for example, 16, 17, 18, 19, or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 19 carbon atoms in the single aliphatic chain backbone, for example, 16, 17, 18, or 19 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 to 18 carbon atoms in the single aliphatic chain backbone, for example, 16, 17, or 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 or 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 16 or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, the two hydrophobic tails each independently contain 18 or 20 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 16 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 17 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 18 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 19 carbon atoms in the single aliphatic chain backbone. In one embodiment, each of the two hydrophobic tails comprises 20 carbon atoms in the single aliphatic chain backbone.

[0309] In one embodiment, each of the at least two (e.g., two) hydrophobic tails is a fatty acid. Non-limiting examples of at least two hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain backbone include octadecylamine, palmitic acid, stearic acid, arachidic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, and derivatives thereof.

[0310] (i) Linker in lipid-anchored polymer In some embodiments, in the lipid-anchored polymers of the present disclosure, the lipid moiety is covalently attached to the polymer, optionally via a linker (lipid moiety and / or lipid moiety with a linker are collectively referred to as "lipid linker" or "linker-lipid moiety" as used herein). In some embodiments, the linker in the lipid-anchored polymers of the present disclosure is a glycerol linker, a phosphate linker, an ether linker, an amide linker, an amine linker, a peptide linker, a phosphoethanolamine linker, a phosphocholine linker, or any combination thereof. In some embodiments, the linker in the lipid-anchored polymers in the LNPs of the present disclosure is a glycerol linker. Thus, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a glycerolipid, which comprises glycerol as a linker and one or more of two lipid moieties described above, e.g., distearoyl-rac-glycerol (DSG).

[0311] In some embodiments, the linker in the lipid-anchored polymer in the LNPs of the present disclosure is a phosphate linker. Thus, in some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is a phospholipid, which comprises a phosphate group as a linker and one or more lipid moieties as described above.

[0312] In some embodiments, the lipid-anchored polymer in the LNPs of the present disclosure is both a glycerolipid and a phospholipid, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0313] In some embodiments, the first lipid-anchored polymer is selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2-oleo ...-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (DSPE and a linker lipid moiety (i.e., having one or more hydrophobic tails containing 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing.

[0314] As used herein, the term "derivative," when used in reference to a linker lipid moiety, means a linker lipid moiety that contains one or more of the following modifications: a) if present, the phosphatidylethanolamine (PE) head group has been modified to convert the amino group to a methylamino or dimethylamino group; b) the modified linker lipid moiety contains one or more additional functional groups or moieties, e.g., -OH, -OCH3, -NH2, maleimide, azide, or cyclooctyne, such as dibenzocyclooctyne (DBCO).

[0315] In one embodiment, the first lipid-anchored polymer comprises a linker lipid moiety (i.e., having one or more hydrophobic tails comprising 16 to 22 carbon atoms in a single aliphatic chain) selected from the group consisting of DOPE, DSPE, DSG, DODA, DPG, derivatives thereof, and combinations of any of the foregoing.

[0316] (ii) Polymer in lipid-anchored polymer In some embodiments, the polymer included in the lipid-anchored polymer is selected from the group consisting of polyethylene (PE), polypropylene (PP), polyethylene glycol (PEG), polyglycerol (PG), polyvinyl alcohol (PVOH), polysarcosine (pSar), and combinations thereof. In one embodiment, the polymer is selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polysarcosine (pSar), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), and combinations thereof. In one embodiment, the polymer is polyethylene glycol (PEG).

[0317] In one embodiment, the polymer is polyethylene glycol (PEG) or a PEG derivative. In another embodiment, the polymer is polyglycerol (PG) or a PG derivative. In yet another embodiment, the polymer is polysarcosine (pSar). In yet another embodiment, the polymer is poly(2-methacryloyloxyethylphosphorylcholine) (PMPC).

[0318] In some embodiments, the polymer in the lipid-anchored polymer has a molecular weight of about 5000 Da or less, e.g., about 4500 Da or less, about 4000 Da or less, about 3500 Da or less, about 3200 Da or less, about 3000 Da or less, about 2500 Da or less, about 2000 Da or less, about 1500 Da or less, about 1000 Da or less, about 500 Da or less, about 100 Da or less, or about 50 Da or less. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 20 Da to about 100 Da, about 50 Da to about 500 Da, about 500 Da to about 2000 Da, about 1000 Da to about 5000 Da, e.g., about 2000 Da to about 5000 Da, about 1000 Da to about 3000 Da, about 1500 Da to about 2500 Da, about 2000 Da to about 4000 Da, or about 2000 Da to about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 1000 Da, about 1500 Da, about 2000 Da, about 2500 Da, about 3000 Da, about 3200 Da, about 3300 Da, about 3350 Da, about 3400 Da, about 3500 Da, about 4000 Da, about 4500 Da, or about 5000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 2000 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3200 Da to about 3500 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3300 Da. In some embodiments, the polymer in the lipid-anchored polymer has an average molecular weight of about 3350 Da. In some embodiments, the polymers in the lipid-anchored polymer have an average molecular weight of about 3400 Da. In some embodiments, the polymers in the lipid-anchored polymer have an average molecular weight of about 3500 Da.

[0319] (iii) a targeting moiety and a second lipid-anchored polymer In some embodiments, the LNPs of the present disclosure further comprise one or more targeting moieties. The targeting moiety targets the LNP for delivery to a specific cell type or tissue in a subject, such as the liver, bone marrow, spleen, or blood. In some embodiments, the targeting moiety can bind to a specific cell type, such as hepatocytes, T cells, B cells, NK cells, dendritic cells, or the like. In some embodiments, the one or more targeting moieties are conjugated to a second lipid-anchored polymer. In some embodiments, the one or more targeting moieties conjugated to the second lipid-anchored polymer can be an antibody.

[0320] The antibody may be an intact monoclonal or polyclonal antibody, as well as an immunologically active fragment (e.g., Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, a humanized antibody, a genetically engineered single-chain Fv (scFv) molecule, or a chimeric antibody, e.g., an antibody that contains the binding specificity of a murine antibody, but the remainder of which is human in origin. Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art. In one embodiment, the targeting moiety is an antibody or antibody fragment, e.g., an antibody or antibody fragment capable of specifically binding to an antigen present on the surface of a cell. In one embodiment, the antibody or antibody fragment is a monoclonal antibody (mAb), a single-chain variable fragment (scFv), a heavy-chain antibody (hcAb), a nanobody (Nb), a heavy-chain-only immunoglobulin (HC1g), an immunoglobulin neoantigen receptor (IgNAR), a variable domain of an immunoglobulin neoantigen receptor (VNAR), a single-domain antibody, or a variable heavy-chain-only antibody (VHH). In one embodiment, the antibody targeting moiety is an scFv. In another embodiment, the antibody targeting moiety is an IgG. In yet another embodiment, the antibody targeting moiety is a VHH (e.g., a nanobody). In some embodiments, the targeting moiety is an antibody directed against an epitope present on a target cell. In some embodiments, the target cell is selected from the group consisting of a T cell, a B cell, a NK cell, a dendritic cell, a hematopoietic cell, a neuronal cell, and a hepatocyte. In some embodiments, the target cell is a T cell. In some embodiments, the antibody targeting moiety binds to an epitope of the T cell receptor (TCR), CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, CD19, CD21, CD28, or PD-1.

[0321] In some other embodiments, the targeting moiety is a ligand (e.g., an oligosaccharide) capable of binding to a receptor present on a target cell. In one embodiment, the targeting moiety is capable of binding to the asialoglycoprotein receptor (ASGPR), i.e., a hepatocyte-specific ASGPR. In one embodiment, the targeting moiety comprises an N-acetylgalactosamine molecule (GalNAc) or a GalNAc derivative thereof. As used herein, "GalNAc derivative" refers to a modified GalNAc molecule or a conjugate of one or more GalNAc molecules (modified or unmodified) covalently linked to a lipid-anchored polymer, e.g., as defined herein. In one embodiment, the targeting moiety is a triantennary or trivalent GalNAc conjugate (i.e., GalNAc3), which is a ligand conjugate having three GalNAc molecules or three GalNAc derivatives. In one embodiment, the targeting moiety is a triantennary GalNAc represented by the following structural formula: [ka]

[0322] In one embodiment, the targeting moiety is a tetraantennary GalNAc conjugate. In one embodiment, the targeting moiety is a tetraantennary or tetravalent GalNAc conjugate (i.e., GalNAc4), which is a ligand having four GalNAc molecules or four GalNAc derivatives.

[0323] In some other embodiments, the targeting moiety is a protein or peptide ligand of a receptor present on the target cell. In one embodiment, the targeting moiety can bind to the low-density lipoprotein receptor (LDLR), e.g., the hepatocyte-specific LDLR. In one embodiment, the targeting moiety comprises an apolipoprotein E (ApoE) protein, an ApoE polypeptide (or peptide), an apolipoprotein B (ApoB) protein, an ApoB polypeptide (or peptide), a fragment of any of the foregoing, or a derivative of any of the foregoing. In one embodiment, the ApoE polypeptide, ApoB polypeptide, or fragment thereof is an ApoE polypeptide, ApoB polypeptide, or fragment thereof disclosed in International Patent Application Publication No. WO 2022 / 261101, the entire contents of which are incorporated herein by reference. In one embodiment, the ApoE protein is a modified ApoE protein, and the ApoB protein is a modified ApoB protein. In one embodiment, the ApoE protein has the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 1). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence set forth in SEQ ID NO:1.In one embodiment, the ApoE protein has the following amino acid sequence:MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHH The ApoE protein has an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:2. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:2. In one embodiment, the ApoE protein has the following amino acid sequence: It has an amino acid sequence that has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98, or at least about 99% sequence identity to MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVR (SEQ ID NO: 3). In one embodiment, the ApoE protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 3. In one embodiment, the ApoE protein comprises the following amino acid sequence: MKVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVSGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRHHHHHHGGSSGSGC (SEQ ID NO: 4) The ApoE protein has an amino acid sequence that shares about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the ApoE protein. In one embodiment, the ApoE protein comprises the amino acid sequence set forth in SEQ ID NO:4. In one embodiment, the ApoE protein consists of the amino acid sequence set forth in SEQ ID NO:4.

[0324] As used herein, the term "sequence identity" refers to the ratio of the number of identical amino acids between two aligned sequences over the aligned length, expressed as a percentage. In some embodiments, the two aligned sequences are identical in length, i.e., have the same number of amino acids.

[0325] In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE protein conjugate in an ApoB protein conjugate, e.g., a conjugate of one or more ApoE and / or ApoB protein molecules (native or modified) or fragments thereof covalently linked to a lipid-anchored polymer as defined herein. In one embodiment, the targeting moiety in the LNPs of the present disclosure is an ApoE polypeptide conjugate in an ApoB polypeptide conjugate, e.g., a conjugate of one or more ApoE and / or ApoB polypeptide molecules or fragments thereof covalently linked to a lipid-anchored polymer as defined herein.

[0326] In one embodiment, an LNP of the present disclosure comprises a second lipid-anchored polymer, wherein a targeting moiety (e.g., mAb, IgG, scFv, VHH, GalNAc, ApoE protein or peptide, ApoB protein or peptide) as defined herein is conjugated to the second lipid-anchored polymer. The second lipid-anchored polymer comprises a C -linker covalently attached to the polymer via a linker. 18 -C 22In one embodiment, the second lipid-anchored polymer is structurally similar to the first lipid-anchored polymer in that it also comprises a lipid moiety comprising a hydrophobic fatty acid tail having a single aliphatic chain backbone of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielaidoyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl-2 -oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), derivatives thereof, and combinations of any of the foregoing. In one embodiment, the second lipid-anchored polymer comprises a lipid linker moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof, and combinations of any of the foregoing.

[0327] The lipid-anchored polymer of the present disclosure may also contain a reactive species. In some embodiments, the reactive species is conjugated to the polymer in the lipid-anchored polymer. The reactive species present in the lipid-anchored polymer of the present disclosure can be used, for example, for conjugation to a targeting moiety functionalized with a complementary reactive species, i.e., a reactive species capable of reacting with the reactive species contained in the lipid-anchored polymer of the present disclosure. In some embodiments, the reactive species conjugated to the lipid-anchored polymer of the present disclosure can be a reagent selected from the group consisting of a thiol reagent, a maleimide reagent, or a click chemistry reagent, for example, an alkyne reagent such as a dibenzocyclooctyne (DBCO) reagent, a transcyclooctene (TCO) reagent, a tetrazine (TZ) reagent, and an azide (AZ) reagent.

[0328] In one embodiment, an antibody or fragment thereof, e.g., an IgG, scFv, VHH, is covalently linked to a lipid-anchored polymer (e.g., a second lipid-anchored polymer) via strain-promoted alkyne azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide, DODA-PG46-azide), and a dibenzocyclooctyne (DBCO)-functionalized scFv, VHH, IgG, or fragment thereof.

[0329] In an exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka]

[0330] In another exemplary embodiment, the second lipid-anchored polymer conjugated to the targeting moiety is represented by the following structure: [ka]

[0331] In one embodiment, the ApoE protein, ApoB protein, ApoE polypeptide, ApoB polypeptide, or fragment thereof is covalently linked to a lipid-anchored polymer (e.g., a second lipid-anchored polymer) via strain-promoted alkyne-azide cycloaddition (SPAAC) chemistry, for example, via an azide-modified lipid-anchored polymer (e.g., DSG-PEG2000-azide, DSPE-PEG2000-azide, DSG-PEG3400-azide, DSPE-PEG3400-azide, DSG-PEG5000-azide, DSPE-PEG5000-azide, DODA-PG-azide), and a dibenzocyclooctyne (DBCO)-functionalized ApoE protein, ApoB protein, ApoE protein, ApoB protein, or fragment thereof.

[0332] In some embodiments, LNPs of the present disclosure can include a first lipid-anchored polymer and a second lipid-anchored polymer. For example, LNPs of the present disclosure can include a first lipid-anchored polymer that does not include a targeting moiety and a second type of lipid-anchored polymer that includes a targeting moiety, such as an scFv, VHH, GalNAc, ApoE protein / peptide, or ApoB protein / peptide. For example, LNPs of the present disclosure can include DSG-PEG2000 modified to include an additional OCH3 group (DSG-PEG2000-OMe) as the first lipid-anchored polymer and DSPE-PEG2000-scFv as the second lipid-anchored polymer.

[0333] In one particular embodiment, the first lipid-anchored polymer is a polymer-conjugated lipid of the present disclosure, e.g., DODA-PG34, DODA-PG45, DODA-PG46, or DODA-PG58. For example, an LNP of the present disclosure may include DODA-PG45 as the first lipid-anchored polymer and DSPE-PEG2000-scFv as the second lipid-anchored polymer.

[0334] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety. In some embodiments, the second lipid-anchored polymer comprises a lipid linker moiety selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and derivatives thereof. In some embodiments, the first lipid-anchored polymer is any lipid-anchored polymer described above.

[0335] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, where the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer have the same lipid linker but different hydrophilic polymers.

[0336] In some embodiments, an LNP of the present disclosure can comprise a first lipid-anchored polymer and a second lipid-anchored polymer, where the second lipid-anchored polymer comprises a targeting moiety, and the first lipid-anchored polymer and the second lipid-anchored polymer differ in their lipid linkers, as shown below: DSG-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DSPE-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DPG-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DPG-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), and DPG-PEG (first lipid-anchored polymer) and DODA-PG (second lipid-anchored polymer).

[0337] In some embodiments, an LNP of the present disclosure may comprise a first lipid-anchored polymer and a second lipid-anchored polymer, wherein the second lipid-anchored polymer comprises a targeting moiety, and wherein the first lipid-anchored polymer and the second lipid-anchored polymer are the same lipid-anchored polymer and are selected from one of the following combinations: DSG-PEG (first lipid-anchored polymer) and DSG-PEG (second lipid-anchored polymer), DSPE-PEG (first lipid-anchored polymer) and DSPE-PEG (second lipid-anchored polymer), DODA-PG (first lipid-anchored polymer) and DODA-PG (second lipid-anchored polymer), and DPG-PEG (first lipid-anchored polymer) and DPG-PEG (second lipid-anchored polymer).

[0338] In some embodiments, the targeting moiety is conjugated to a DSPE anchor polymer, hi some embodiments, the DSPE anchor polymer is DSPE-PEG or a derivative thereof.

[0339] In some embodiments, the targeting moiety is conjugated to a DSG anchor polymer, hi some embodiments, the DSG anchor polymer is DSG-PEG or a derivative thereof.

[0340] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-IgG.

[0341] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-VHH.

[0342] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-scFv.

[0343] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DODA-PG-VHH.

[0344] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG46 (i.e., a polyglycerol with an average of 46 glycerol repeat units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and bis-DODA-PG46 (e.g., d18:1 / 2:0 or d14:1 / 2:0). In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG46.

[0345] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34 (i.e., a polyglycerol with an average of 34 glycerol units). In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DODA-PG34.

[0346] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-VHH.

[0347] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG46, and DODA-PG46-scFv.

[0348] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DODA-PG-VHH.

[0349] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DODA-PG-VHH.

[0350] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OMe. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-VHH.

[0351] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, and DSG-PEG2000-OH. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-VHH. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-VHH.

[0352] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OMe, and DSPE-PEG2000-scFv.

[0353] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DSG-PEG2000-OH, and DSPE-PEG2000-scFv.

[0354] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, bis-DSG-PEG2000, and DSPE-PEG2000-scFv.

[0355] In some embodiments, LNPs provided by the present disclosure comprise a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG, and DSPE-PEG-scFv. In some embodiments, LNPs provided by the present disclosure consist essentially of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG45, and DSPE-PEG2000-scFv. In some embodiments, LNPs provided by the present disclosure consist of a therapeutic nucleic acid (TNA), an ionizable lipid, a helper lipid (e.g., DSPC, DOPE, ceramide), cholesterol, DODA-PG45, and DSPE-PEG2000-scFv.

[0356] In some embodiments, the lipid-anchored polymer (the first lipid-anchored polymer and the second lipid-anchored polymer in combination) comprises about 0.1 mol % to about 20 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 0.5 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 1 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 2 mol % to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises more than about 2 mol % (e.g., 2.1 mol %, 2.2 mol %, 2.3 mol %, 2.4 mol %, 2.5 mol %, 2.6 mol %, 2.7 mol %, 2.8 mol %, 2.9 mol %, 3.0 mol %) to about 10 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol % to about 8 mol % of the total lipid present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% to about 7 mol% present in the LNP. In some embodiments, the lipid-anchored polymer comprises about 3 mol% to about...

Claims

1. A stealth lipid nanoparticle (LNP), comprising: (a) a therapeutic nucleic acid (TNA); (b) an ionizable lipid; and (c) a sterol; (d) a first lipid-anchored polymer; and (e) a second lipid-anchored polymer, optionally comprising a reactive moiety; A stealth lipid nanoparticle (LNP), wherein the first lipid-anchored polymer and the second lipid-anchored polymer each comprise a lipid linker and a hydrophilic polymer.

2. The stealth LNP of claim 1, wherein the reactive portion of the second lipid-anchored polymer is located on the exterior of the LNP.

3. The stealth LNP of claim 1 or 2, further comprising a covalent linker between the second lipid-anchored polymer and the reactive moiety.

4. The stealth LNP of any one of claims 1 to 3, wherein the reactive moiety is maleimide or thiol.

5. The stealth LNP of any one of claims 1 to 4, wherein the reactive moiety is maleimide.

6. The stealth LNP of any one of claims 1 to 4, wherein the reactive moiety is a thiol.

7. The stealth LNP of any one of claims 1 to 3, wherein the reactive moiety is a click chemistry reagent.

8. The stealth LNP of any one of claims 1 to 3 or 7, wherein the reactive moiety is azide or DBCO.

9. The stealth LNP of any one of claims 1-3 or 7-8, wherein the reactive moiety is an azide.

10. The stealth LNP of any one of claims 1-3 or 7-8, wherein the reactive moiety is DBCO.

11. A stealth lipid nanoparticle (LNP), comprising: (a) a therapeutic nucleic acid (TNA); (b) an ionizable lipid; and (c) a sterol; (d) a first lipid-anchored polymer; and (e) a second lipid-anchored polymer, wherein the second lipid-anchored polymer is conjugated to a targeting moiety; A stealth lipid nanoparticle (LNP), wherein the first lipid-anchored polymer and the second lipid-anchored polymer each comprise a lipid linker and a hydrophilic polymer.

12. The stealth LNP of claim 11, wherein the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety.

13. 13. The stealth LNP of claim 11 or 12, wherein the targeting moiety is selected from the group consisting of proteins, nucleic acids, and sugars.

14. The stealth LNP of any one of claims 11 to 13, wherein the targeting moiety is an antibody, an antibody fragment, or an antibody derivative.

15. 15. The stealth LNP of claim 14, wherein the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single domain antibody, a single-chain antibody, and a variable heavy chain-only antibody (VHH).

16. The stealth LNP of any one of claims 13 to 15, wherein the antibody, antibody fragment, or antibody derivative is an scFv.

17. The stealth LNP of any one of claims 13 to 15, wherein the antibody, antibody fragment, or antibody derivative is a VHH.

18. The stealth LNP of claim 17, wherein the VHH is a nanobody.

19. The stealth LNP of any one of claims 11 to 18, wherein the targeting moiety is located on the exterior of the LNP.

20. The stealth LNP of any one of claims 11 to 13, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

21. The stealth LNP of any one of claims 11 to 13, wherein the targeting moiety is an aptamer.

22. The stealth LNP of any one of claims 11 to 19 or 21, wherein the targeting moiety specifically binds to a T cell antigen.

23. 23. The stealth LNP of any one of claims 11-19 or 21-22, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR.

24. The stealth LNP of any one of claims 11 to 19 or 21 to 23, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.

25. The stealth LNP of any one of claims 11 to 24, further comprising a linker between the second lipid-anchored polymer and the targeting moiety.

26. The stealth LNP of any one of claims 1 to 25, wherein the first lipid linker and the second lipid linker are each independently selected from the group consisting of non-ester-containing linkers and ester-containing linkers.

27. The stealth LNP of any one of claims 1 to 26, wherein the ester-containing linker is selected from the group consisting of an amide linker and a carbamate linker.

28. The stealth LNP of any one of claims 11 to 27, wherein the targeting moiety is conjugated to the second lipid-anchored polymer via maleimide conjugation.

29. The stealth LNP of any one of claims 11 to 28, wherein the targeting moiety is conjugated to the second lipid-anchored polymer via click chemistry.

30. 30. The stealth LNP of any one of claims 1-29, wherein the sterol is selected from the group consisting of cholesterol, beta-sitosterol, stigmasterol, beta-sitostanol, campesterol, brassicasterol, derivatives thereof, and combinations thereof.

31. The stealth LNP of any one of claims 1 to 30, wherein the sterol is cholesterol.

32. The stealth LNP of any one of claims 1 to 30, wherein the sterol is beta-sitosterol.

33. The ionizable lipid is selected from the group consisting of 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl DLin-[1,3]-dioxolane (DLin-K-DMA), DLin-MC3-DMA, N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 1,2-dilauro ... Phosphorus (DLEPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)aminolbutylcarboxamidoethyl-3,4-di[oleyloxy]-benzamide (MVL5), dioctadecylamido-glycylspermine (DOGS), 3b-[ N-(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol), dioctadecyldimethylammonium bromide (DDAB), Saint lipids (e.g., SAINT-2, N-methyl-4-(dioleyl)methylpyridinium), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1,The stealth LNP of any one of claims 1 to 21, wherein the LNP is selected from the group consisting of 2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acid (DILA2) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), and 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In some variations, the condensing agent, e.g., cationic lipid, is selected from the group consisting of, for example, dioctadecyldimethylammonium bromide (DDAB), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2 dimethylaminoethyl)-[1,31-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1, Lipids such as 2-dioleyloxy-3-dimethylaminopropane (DODMA), morpholino cholesterol (Mo-CHOL), (R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride (DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G), and (R)-N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride (DOTAPen), SMA102, L369, LP01, "SS-cleavable lipids," and mixtures thereof.

34. The stealth LNP of any one of claims 1 to 33, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a lipid comprising at least one hydrophobic tail.

35. The stealth LNP of any one of claims 1 to 34, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a lipid comprising at least two hydrophobic tails.

36. Each hydrophobic tail has at least 18 carbon atoms (C １８ 36. The stealth LNP of claim 34 or 35, comprising a carbon chain having the formula:

37. Each hydrophobic tail is 18 to 22 carbon atoms (C １８ -C ２２ 37. The stealth LNP of any one of claims 34 to 36, comprising a carbon chain having the formula:

38. Each hydrophobic tail has 18 carbon atoms (C １８ 38. The stealth LNP of any one of claims 34 to 37, comprising a carbon chain having the formula:

39. The first lipid linker and the second lipid linker are each independently selected from the group consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dielideyl-sn-phosphatidylethanolamine (DEPE), 1-stearoyl- 39. The stealth LNP of any one of claims 1-38, wherein the LNP is selected from the group consisting of 2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 18-1-trans PE, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), and dioctadecylamine (DODA), distearoyl-rac-glycerol (DSG), 1,2-dipalmitoyl-rac-glycerol (DPG), and combinations and derivatives thereof.

40. The stealth LNP of any one of claims 1 to 39, wherein the first lipid linker and the second lipid linker are each independently selected from the group consisting of DSPE, DSG, DODA, DPG, DOPE, and combinations and derivatives thereof.

41. The stealth LNP of any one of claims 1 to 40, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are each independently DSPE, DODA, DSG, or a combination thereof.

42. 42. The stealth LNP of any one of claims 1-41, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise a polymer selected from the group consisting of polyethylene glycol (PEG), polyglycerol (PG), polyoxazoline (POZ), poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), polyamide, and combinations thereof.

43. 43. The stealth LNP of claim 42, wherein the polymer is PEG.

44. The stealth LNP of claim 42 or 43, wherein the PEG is selected from the group consisting of PEG2000, PEG2000Ome, and PEG2000-OH.

45. 44. The stealth LNP of claim 43, wherein the polymer is polyglycerol (PG).

46. The stealth LNP of claim 29 or 32, wherein the PG comprises at least 5 to 60 glycerol units.

47. 47. The stealth LNP of any one of claims 1 to 46, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each independently comprise DSPE, DODA, DSG, or a combination thereof.

48. 48. The stealth LNP of any one of claims 1-47, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are each independently DSPE-PEG, DODA-PG, DSPE-PG, DODA-PEG, DSG-PEG, DSG-PG, or a combination thereof.

49. The stealth LNP of any one of claims 1 to 48, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each comprise a different lipid linker.

50. The stealth LNP of any one of claims 1 to 48, wherein the first lipid-anchored polymer and the second lipid-anchored polymer each comprise the same lipid linker.

51. The stealth LNP of any one of claims 1 to 50, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are different.

52. The stealth LNP of any one of claims 1 to 50, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are the same.

53. The stealth LNP of any one of claims 1 to 35, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are both DSPE-PEG.

54. The stealth LNP of any one of claims 1 to 34 or 36, wherein the first lipid-anchored polymer and the second lipid-anchored polymer are both DODA-PG.

55. The stealth LNP of any one of claims 1 to 54, further comprising a helper lipid.

56. The helper lipid may be distearoyl-sn-glycero-phosphoethanolamine (DSPE), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE ), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), dierucoyl phosphatidylcholine (DEPC), palmitoyl oleyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,56. The stealth LNP of claim 55, selected from the group consisting of 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, DODA, ceramide, and derivatives and combinations thereof.

57. The stealth LNP of claim 55 or 56, wherein the helper lipid is DSPC.

58. The stealth LNP of any one of claims 1 to 57, wherein the ionizable lipid is present in a molar percentage of about 30% to about 80%.

59. 59. The stealth LNP of any one of claims 1-58, wherein the sterol is present in a molar percentage of about 20% to about 50%.

60. 60. The stealth LNP of any one of claims 1-59, wherein the sterol is present in a molar percentage of about 35% to about 40%.

61. 61. The stealth LNP of any one of claims 1 to 60, wherein the first lipid-anchored polymer and the second anchored polymer are present in a combined molar percentage of about 1% to about 8%.

62. The stealth LNP of any one of claims 1 to 61, wherein the first lipid-anchored polymer and the second anchored polymer are present in a combined molar percentage of about 2% to about 5%.

63. 63. The stealth LNP of any one of claims 1 to 62, wherein the first lipid-anchored polymer and the second anchored polymer are present in a combined molar percentage of about 3%.

64. The stealth LNP of any one of claims 1 to 63, wherein the first lipid-anchored polymer is present in a molar percentage of about 1% to about 7%.

65. The stealth LNP of any one of claims 1 to 64, wherein the first lipid-anchored polymer is present in a molar percentage of about 1.5% to about 5%.

66. 66. The stealth LNP of any one of claims 1 to 65, wherein the first lipid-anchored polymer is present in a molar percentage of about 2% to about 3%.

67. 67. The stealth LNP of any one of claims 1 to 66, wherein the first lipid-anchored polymer is present in a molar percentage of about 2% to about 3%.

68. The stealth LNP of any one of claims 1 to 67, wherein the first lipid-anchored polymer is present at a molar percentage of about 2.5%.

69. The stealth LNP of any one of claims 1 to 68, wherein the second lipid-anchored polymer is present in a molar percentage of about 0.25% to about 1%.

70. 70. The stealth LNP of any one of claims 1 to 69, wherein the second lipid-anchored polymer is present in a molar percentage of about 0.35% to about 0.75%.

71. The stealth LNP of any one of claims 1 to 70, wherein the second lipid-anchored polymer is present at a molar percentage of about 0.5%.

72. The stealth LNP of any one of claims 55 to 71, wherein the helper lipid is present in a molar percentage of about 2% to about 20%.

73. The stealth LNP of any one of claims 55 to 72, wherein the helper lipid is present at a molar percentage of about 10%.

74. The stealth LNP of any one of claims 1 to 73, further comprising an immunosuppressant.

75. The stealth LNP of any one of claims 1 to 74, wherein the nanoparticle has a total lipid to TNA ratio of about 10:1 to about 40:

1.

76. The stealth LNP of any one of claims 1 to 75, wherein the LNP has a diameter of about 40 nm to about 120 nm.

77. The stealth LNP of any one of claims 1 to 76, wherein the LNP has a diameter of less than about 100 nm.

78. The stealth LNP of any one of claims 1 to 77, wherein the LNP has a diameter of about 60 nm to about 80 nm.

79. The stealth LNP of any one of claims 1 to 78, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 40 nm to about 120 nm.

80. The stealth LNP of any one of claims 1 to 79, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of less than about 100 nm.

81. The stealth LNP of any one of claims 1 to 80, wherein the LNP is present in an LNP composition comprising a plurality of LNPs having an average diameter of about 60 nm to about 80 nm.

82. The stealth LNP of any one of claims 1 to 81, wherein the TNA is selected from the group consisting of RNA, DNA, and derivatives and analogs thereof.

83. The stealth LNP of any one of claims 1 to 82, wherein the TNA encodes a therapeutic gene and / or a therapeutic protein.

84. The stealth LNP of any one of claims 1 to 83, wherein the TNA is selected from the group consisting of mRNA, siRNA, synthetic ribozyme, antisense RNA, and gRNA.

85. The stealth LNP of any one of claims 1 to 84, wherein the TNA is mRNA.

86. The stealth LNP of any one of claims 1 to 83, wherein the TNA is selected from the group consisting of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA).

87. The stealth LNP of any one of claims 1 to 83 or 86, wherein the TNA is ssDNA.

88. The stealth LNP of any one of claims 1 to 83 or 86 to 87, wherein the TNA is linear ssDNA.

89. The stealth LNP of any one of claims 1 to 83 or 86, wherein the TNA is dsDNA.

90. The stealth LNP of any one of claims 1 to 83, 86, or 89, wherein the TNA is a capsid-free non-viral DNA vector with covalently closed ends (ceDNA vector).

91. The stealth LNP of any one of claims 1 to 90, wherein the TNA encodes a chimeric antigen receptor (CAR).

92. The stealth LNP of claim 91, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain.

93. The stealth LNP of claim 91 or 92, wherein the signaling domain is a CD3 zeta signaling domain.

94. The stealth LNP of any one of claims 91 to 93, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

95. The stealth LNP of claim 94, wherein the antigen-binding fragment is a Fab, a Fab', a scFv, or a VHH.

96. The stealth LNP of any one of claims 92 to 95, wherein the antigen-binding domain binds to a tumor antigen.

97. The stealth LNP of claim 96, wherein the tumor antigen is associated with a hematological malignancy.

98. The stealth LNP of claim 97, wherein the tumor antigen is associated with a solid tumor.

99. The stealth LNP of any one of claims 92 to 98, wherein the costimulatory signaling region comprises the intracellular domain of a costimulatory molecule selected from the group consisting of CD13, CD19, CD21, CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and any combination thereof.

100. The stealth LNP of any one of claims 1 to 99, wherein the TNA is synthetically produced in a cell-free environment.

101. The stealth LNP of any one of claims 1 to 100, wherein the TNA encodes a therapeutic gene and / or a therapeutic protein.

102. A cell comprising the stealth LNP of any one of claims 1 to 101.

103. 102. The cell of claim 101, wherein the cell is in vitro, ex vivo, or in vivo.

104. 104. The cell of claim 102 or 103, wherein the cell is a T cell.

105. The cell of any one of claims 102 to 104, wherein the cell is an autologous T cell.

106. The cell of any one of claims 102 to 105, wherein the cell is an allogeneic T cell.

107. A pharmaceutical composition comprising a stealth LNP described in any one of claims 1 to 101 or a cell described in any one of claims 102 to 106.

108. 108. The pharmaceutical composition of claim 107, further comprising a pharmaceutically acceptable excipient or carrier.

109. 109. The pharmaceutical composition of claim 107 or 108, further comprising an immunosuppressant.

110. The pharmaceutical composition of any one of claims 107 to 109, further comprising a tyrosine kinase inhibitor (TKI).

111. 111. The pharmaceutical composition of claim 110, wherein the tyrosine kinase inhibitor is a pharmaceutically acceptable salt of the TKI.

112. A method for treating a disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of a stealth LNP described in any one of claims 1 to 101, a cell described in any one of claims 102 to 106, and / or a pharmaceutical composition described in any one of claims 107 to 111.

113. 113. The method of claim 112, wherein the disease or disorder is a genetic disease or disorder.

114. The genetic disease or disorder may be sickle cell anemia, melanoma, hemophilia A (clotting factor VIII (FVIII) deficiency) and hemophilia B (clotting factor IX (FIX) deficiency), cystic fibrosis (CFTR), familial hypercholesterolemia (LDL receptor deficiency), hepatoblastoma, Wilson's disease, phenylketonuria (PKU), congenital hepatic porphyria, inherited liver metabolic disorders, Lesch-Nyhan syndrome, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS I), Scheie syndrome (MPS I S), Hurler-Scheie syndrome (MPS I H-S), Hunter syndrome (MPS II), Sanfilippo types A, B, C, and D (MPS III MPS A, B, C, and D), Morquio syndrome types A and B (MPS IVA and MPS IVB), Maroteaux-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease types A / B, C1, and C2, Fabry disease, Schindler disease, GM2-gangliosidosis type II (Sandhoff disease), Tay-Sachs disease, metachromatic leukodystrophy, Krabbe disease, mucolipidosis types I, II / III, and IV, sialidosis types I and II, glycogen storage disease types I and II (Pompe disease), Gaucher disease types I and II Type III and III, cystinosis, Batten disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidoses, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, 114. The method of claim 112 or 113, wherein the inflammatory bowel disease is selected from the group consisting of: Tony's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, age-related macular degeneration (AMD), alpha-1 antitrypsin deficiency, progressive familial intrahepatic cholestasis (PFIC) type I (ATP8B1 deficiency), type II (ABCB11), type III (ABCB4), or type IV (TJP2), and cathepsin A deficiency.

115. The method of any one of claims 112 to 114, wherein the disease or disorder is hemophilia A.

116. The method of any one of claims 112 to 114, wherein the disease or disorder is hemophilia B.

117. 115. The method of any one of claims 112 to 114, wherein the disease or disorder is phenylketonuria (PKU).

118. 115. The method of any one of claims 112 to 114, wherein the disease or disorder is Wilson's disease.

119. 115. The method of any one of claims 112 to 114, wherein the disease or disorder is Gaucher disease types I, II, and III.

120. 115. The method of any one of claims 112 to 114, wherein the disease or disorder is Stargardt's macular dystrophy.

121. The method of any one of claims 112 to 114, wherein the disease or disorder is LCA10.

122. 115. The method of any one of claims 112 to 114, wherein the disease or disorder is Usher syndrome.

123. The method of any one of claims 112 to 114, wherein the disease or disorder is wet AMD.

124. A method for delivering a therapeutic nucleic acid (TNA) to a subject, comprising administering to the subject a therapeutically effective amount of a stealth LNP described in any one of claims 1 to 101, a cell described in any one of claims 102 to 106, and / or a pharmaceutical composition described in any one of claims 107 to 111.

125. A method for delivering a therapeutic gene and / or a therapeutic protein, the therapeutic gene and / or the therapeutic protein being encoded by a therapeutic nucleic acid (TNA), to a cell, the method comprising contacting the cell with a stealth LNP described in any one of claims 1 to 101 and / or a pharmaceutical composition described in any one of claims 107 to 111 in a subject, thereby delivering the therapeutic gene and / or the therapeutic protein to the cell.

126. A method for delivering a therapeutic gene to the nucleus of a cell, comprising contacting the cell with a stealth LNP described in any one of claims 1 to 101 and / or a pharmaceutical composition described in any one of claims 107 to 111 in a subject, thereby delivering the therapeutic gene and / or therapeutic protein to the nucleus of the cell.

127. 127. The method of claim 125 or 126, wherein the cell is in vitro.

128. 127. The method of claim 125 or 126, wherein the cell is in vivo.

129. 127. The method of claim 125 or 126, wherein the cells are ex vivo.

130. A method for providing anti-tumor immunity to a subject, the method comprising administering to the subject a stealth LNP described in any one of claims 1 to 101, a cell described in any one of claims 102 to 106, and / or a pharmaceutical composition described in claims 107 to 111, thereby providing anti-tumor immunity to the subject.

131. A method of treating a subject having a disease, disorder, or condition associated with elevated expression of a tumor antigen, the method comprising administering to the subject a stealth LNP described in any one of claims 1 to 101, a cell described in any one of claims 102 to 106, or a pharmaceutical composition described in any one of claims 106 to 111, thereby treating the subject.

132. 130. The method of any one of claims 125 to 129, wherein the cell is a T cell.

133. 133. The method of any one of claims 125-129 or 132, wherein the cells are autologous T cells.

134. 133. The method of any one of claims 125-129 or 132, wherein the cells are allogeneic T cells.

135. 132. The method of any one of claims 112-124 or 130-131, wherein the subject is a human.

136. 1. A method for generating stealth LNPs comprising a targeting moiety, comprising: (a) providing a stealth LNP according to any one of claims 1 to 10, wherein the second lipid-anchored polymer comprises a first reactive moiety; (b) providing a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety are capable of reacting to form a covalent bond; (c) contacting the stealth LNP of (a) with the targeting moiety of (b) under conditions sufficient to allow a reaction between the first reactive moiety and the second reactive moiety; Thereby generating stealth LNPs containing a targeting moiety.

137. 137. The method of claim 136, wherein the first reactive moiety is a maleimide and the second reactive moiety is a thiol.

138. 137. The method of claim 136, wherein the first reactive moiety is a thiol and the second reactive moiety is a maleimide.

139. 137. The method of claim 136, wherein the first reactive moiety and the second reactive moiety are click chemistry reagents.

140. 140. The method of claim 136 or 139, wherein the first reactive moiety is azide and the second reactive moiety is DBCO.

141. 140. The method of claim 136 or 139, wherein the first reactive moiety is DBCO and the second reactive moiety is azide.

142. 142. The method of any one of claims 136 to 141, wherein the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety.

143. 143. The method of any one of claims 136 to 142, wherein the targeting moiety is selected from the group consisting of proteins, nucleic acids, and sugars.

144. 144. The method of any one of claims 136 to 143, wherein the targeting moiety is an antibody, an antibody fragment, or an antibody derivative.

145. 145. The method of claim 144, wherein the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single domain antibody, a single chain antibody, and a VHH.

146. 146. The method of claim 144 or 145, wherein the antibody, antibody fragment, or antibody derivative is an scFv.

147. The method of claim 144 or 145, wherein the antibody, antibody fragment, or antibody derivative is a VHH.

148. The method of claim 147, wherein the VHH is a nanobody.

149. 144. The method of any one of claims 136-143, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

150. 144. The method of any one of claims 136 to 143, wherein the targeting moiety is an aptamer.

151. 151. The method of any one of claims 136-148 or 150, wherein the targeting moiety specifically binds to a T cell antigen.

152. 152. The method of any one of claims 136-148 or 150-151, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR.

153. 153. The method of any one of claims 136-148 or 150-152, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.

154. A kit for the preparation of targeted stealth LNPs, comprising: (a) the stealth LNP of any one of claims 1 to 10, wherein the second lipid-anchored polymer comprises a first reactive moiety; (b) instructions for producing a targeted stealth LNP by contacting the stealth LNP of (a) with a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety are capable of reacting to form a covalent bond.

155. 1. A kit for the production of targeted stealth LNPs, comprising: (a) the stealth LNP of any one of claims 1 to 10, wherein the second lipid-anchored polymer comprises a first reactive moiety; (b) a targeting moiety comprising a second reactive moiety, wherein the first reactive moiety and the second reactive moiety can react with each other to form a covalent bond; (c) instructions for producing a targeted stealth LNP by contacting the stealth LNP of (a) with the targeting moiety of (b).

156. 156. The kit of claim 154 or 155, wherein the first reactive moiety is a maleimide and the second reactive moiety is a thiol.

157. 156. The kit of claim 154 or 155, wherein the first reactive moiety is a thiol and the second reactive moiety is a maleimide.

158. 156. The kit of claim 154 or 155, wherein the first reactive moiety and the second reactive moiety are click chemistry reagents.

159. 159. The kit of claims 154-155 or 158, wherein the first reactive moiety is azide and the second reactive moiety is DBCO.

160. 159. The kit of claims 154-155 or 158, wherein the first reactive moiety is DBCO and the second reactive moiety is azide.

161. 161. The kit of any one of claims 154 to 160, wherein the targeting moiety is a tissue-specific and / or cell-type-specific targeting moiety.

162. 162. The kit of any one of claims 154 to 161, wherein the targeting moiety is selected from the group consisting of proteins, nucleic acids, and sugars.

163. 163. The kit of any one of claims 154 to 162, wherein the targeting moiety is an antibody, an antibody fragment, or an antibody derivative.

164. 164. The kit of any one of claims 154 to 163, wherein the antibody, antibody fragment, or antibody derivative is selected from the group consisting of a full-length antibody, a Fab, a Fab', a single domain antibody, a single chain antibody, and a VHH.

165. 165. The kit of any one of claims 154 to 164, wherein the antibody, antibody fragment, or antibody derivative is an scFv.

166. 165. The kit of any one of claims 154 to 164, wherein the antibody, antibody fragment, or antibody derivative is a VHH.

167. The kit of claim 166, wherein the VHH is a nanobody.

168. 163. The kit of any one of claims 154-162, wherein the targeting moiety is N-acetylgalactosamine (GalNAc) or a GalNAc derivative.

169. 163. The kit of any one of claims 154 to 162, wherein the targeting moiety is an aptamer.

170. 170. The kit of any one of claims 154-167 or 169, wherein the targeting moiety specifically binds to a T cell antigen.

171. 171. The kit of any one of claims 154-167 or 169-170, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11, PD-1, and TCR.

172. 172. The kit of any one of claims 154-167 or 169-171, wherein the targeting moiety binds to a T cell antigen selected from the group consisting of CD3, CD5, CD6, and CD7.

173. 1. A stealth lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), an ionizable lipid number 87, cholesterol, a first lipid anchor polymer, and a second lipid anchor polymer, wherein the first lipid anchor polymer is DSG-PEG2000-OMe and the second lipid anchor polymer is DSPE-PEG5000-maleimide reactive moiety.

174. The stealth LNP of claim 173, comprising a therapeutic nucleic acid (TNA), about 57.5 mol% of the ionizable lipid number 87, about 39.5 mol% of cholesterol, about 2.5 mol% of the first lipid-anchored polymer, and about 0.5 mol% of the second lipid-anchored polymer.

175. 1. A stealth lipid nanoparticle (LNP) comprising a therapeutic nucleic acid (TNA), an ionizable lipid number 87, DSPC, cholesterol, a first lipid anchor polymer, and a second lipid anchor polymer, wherein the first lipid anchor polymer is DSG-PEG2000-OMe and the second lipid anchor polymer is DSPE-PEG5000-maleimide reactive moiety.

176. The stealth LNP of claim 175, comprising a therapeutic nucleic acid (TNA), about 47.5 mol% of the ionizable lipid number 87, about 10 mol% DSPC, about 39.5 mol% cholesterol, about 2.5 mol% of the first lipid anchor polymer, and about 0.5 mol% of the second lipid anchor polymer.

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