Novel lipids and nanoparticle compositions thereof

Abstract

To provide lipids having the formula (I) and pharmaceutically acceptable salts thereof.SOLUTION: In the formula, R1, R2, a, and b are as defined herein. Also provided herein are lipid nanoparticle (LNP) compositions comprising a lipid having the formula (I) and a capsid-free, non-viral vector (e.g., ceDNA). In one aspect or any of the aspects or embodiments herein, these LNPs can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 000,990, filed March 27, 2020, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application has been submitted electronically in ASCII format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy, created on March 18, 2021, is named 131698_07720_SL.txt and is 417 bytes in size. [Background technology]

[0003] Gene therapy aims to improve clinical outcomes for patients suffering from either inherited or acquired disorders caused by abnormal gene expression profiles. Various types of gene therapy have been developed to deliver therapeutic nucleic acids into patients' cells as drugs to treat diseases.

[0004] Introduction and expression of repair genes into target cells in patients can be achieved through a number of methods, including the use of engineered viral gene delivery vectors, and potentially plasmids, minigenes, oligonucleotides, minicircles, or various closed-end DNA vectors. Among the many available viral vectors (e.g., recombinant retroviruses, lentiviruses, and adenoviruses), recombinant adeno-associated viruses (rAAVs) have been accepted as a versatile and relatively safe vector for gene therapy. However, viral vectors such as adeno-associated vectors are highly immunogenic and can induce humoral and cellular immunity that can impair efficacy, especially upon re-administration.

[0005] Non-viral gene delivery avoids certain disadvantages associated with viral transduction, particularly the humoral and cellular immune responses to the viral structural proteins that form the vector particle, and those resulting from any de novo viral gene expression. Among the non-viral gene delivery techniques is the use of cationic lipids as carriers.

[0006] Ionizable lipids are generally composed of an amine moiety and a lipid moiety, and the cationic amine moiety interacts electrostatically with polyanionic nucleic acids to form positively charged liposomes or lipid membrane structures, thereby facilitating cellular uptake and delivery of nucleic acids into cells.

[0007] Widely used ionizable lipids include cationic lipids such as CLinDMA, DLinDMA (also known as DODAP), and DOTAP. Notably, these lipids have been used to deliver siRNA to the liver, but suffer from suboptimal delivery efficiency along with liver toxicity at high doses. Given the shortcomings of current cationic lipids, it is necessary to provide lipid scaffolds that not only exhibit enhanced efficacy with reduced toxicity, but also improve pharmacokinetics and intracellular dynamics, such as cellular uptake and nucleic acid release from lipid carriers. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, an ionizable lipid having formula (I): [ka] In the formula, R 1 , R 2 Provided herein are lipids, and pharmaceutically acceptable salts thereof, wherein a, a, and b are as defined herein.

[0009] Also provided are pharmaceutical compositions comprising the disclosed ionizable lipids, or pharmaceutically acceptable salts thereof, and a pharmaceutically acceptable carrier.

[0010] Another aspect of the present disclosure relates to a composition comprising a lipid nanoparticle (LNP) comprising an ionizable lipid described herein, or a pharmaceutically acceptable salt thereof, and a nucleic acid. In one embodiment of any of the aspects or embodiments herein, the nucleic acid is encapsulated in the ionizable lipid. In certain embodiments, the nucleic acid is closed-end DNA (ceDNA).

[0011] According to some embodiments of any of the aspects or embodiments herein, the LNP further comprises a sterol. According to some embodiments of any of the aspects or embodiments herein, the sterol can be cholesterol or beta-sitosterol.

[0012] According to some embodiments of any of the aspects or embodiments herein, the cholesterol is present in a molar percentage of about 20% to about 40%, e.g., about 20% to about 35%, about 20% to about 30%, about 20% to about 25%, about 25% to about 35%, about 25% to about 30%, or about 30% to about 35%, and the ionizable lipid is present in a molar percentage of about 80% to about 60%, e.g., about 80% to about 65%, about 80% to about 70%, about 80% to about 75%, about 75% to about 60%, about 75% to about 65%, about 75% to about 70%, about 70% to about 60%, or about 70% to about 60%. According to some embodiments of any of the aspects or embodiments herein, the cholesterol is about 20% to about 40%, e.g., about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%. According to some embodiments of any of the aspects or embodiments herein, cholesterol is present at a molar percentage of about 40% and the ionizable lipid is present at a molar percentage of about 50%.

[0013] According to some embodiments of any of the aspects or embodiments herein, the composition further comprises cholesterol, a PEG-lipid conjugate, and a non-cationic lipid. According to some embodiments of any of the aspects or embodiments herein, the PEG-lipid conjugate is present at about 1.5% to about 3%, e.g., about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, about 2% to about 2.25%, about 2.25% to about 3%, about 2.25% to about 2.75%, or about 2.25% to about 2.5%. According to some embodiments of any of the aspects or embodiments herein, the PEG-lipid conjugate is present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3%. According to some embodiments of any of the aspects or embodiments herein, the cholesterol is present at a molar percentage of about 30% to about 50%, e.g., about 30% to about 45%, about 30% to about 40%, about 30% to about 35%, about 35% to about 50%, about 35% to about 45%, about 35% to about 40%, about 20% to about 40%, about 40% to about 50%, or about 45% to about 50%. According to some embodiments of any of the aspects or embodiments herein, the cholesterol is present in a molar percentage of about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50%.

[0014] According to some embodiments of any of the aspects or embodiments herein, the LNP further comprises a polyethylene glycol (PEG)-lipid. According to some embodiments of any of the aspects or embodiments herein, the PEG-lipid is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). According to some embodiments of any of the aspects or embodiments herein, the LNP further comprises a non-cationic lipid. According to some embodiments of any of the aspects or embodiments herein, the non-cationic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, 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 (DSPC), 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-phosphatidiethanolamine (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 oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,The non-cationic 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, phosphatidicaside, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or a mixture thereof. According to some embodiments of any of the aspects or embodiments herein, the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE).

[0015] According to some embodiments of any of the aspects or embodiments herein, the PEG-lipid conjugate is present at about 1.5% to about 4%, e.g., about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 1.5% to about 2.75%, about 1.5% to about 2.5%, about 1.5% to about 2.25%, about 1.5% to about 2%, about 1.5% to about 1.75%, about 2% to about 3%, about 2% to about 2.75%, about 2% to about 2.5%, or about 2% to about 2.25%. According to some embodiments of any of the aspects or embodiments herein, the PEG-lipid conjugate is present at about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3%. According to some embodiments of any of the aspects or embodiments herein, the ionizable lipid is present at a molar percentage of about 42.5% to about 62.5%. According to some embodiments of any of the aspects or embodiments herein, the ionizable lipid is about 42.5%, about 43%, about 43.5%, about 44%, about 44.5%, about 45%, about 45.5%, about 46%, about 46.5%, 47.5%, about 48%, about 48.5%, about 49%, about 49.5%, about 50%, about 50.5%, about 51%, about 51.5%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about According to some embodiments of any of the aspects or embodiments herein, the non-cationic lipid is present in a molar percentage of about 2.5% to about 12.5%. According to some embodiments of any of the aspects or embodiments herein, the cholesterol is present at a molar percentage of about 40%, the ionizable lipid is present at a molar percentage of about 52.5%, the non-cationic lipid is present at a molar percentage of about 7.5%, and the PEG-lipid is present at about 3%.

[0016] According to some embodiments of any of the aspects or embodiments herein, the LNP composition further comprises dexamethasone palmitate.

[0017] According to some embodiments of any of the aspects or embodiments herein, the LNP has a diameter of about 50 nm to about 110 nm, e.g., about 50 nm to about 100 nm, about 50 nm to about 95 nm, about 50 nm to about 90 nm, about 50 nm to about 85 nm, about 50 nm to about 80 nm, about 50 nm to about 75 nm, about 50 nm to about 70 nm, about 50 nm to about 65 nm, about 50 nm to about 60 nm, about 50 nm to about 55 nm, about 60 nm to about 110 nm, about 60 nm to about 100 nm, about 60 nm to about 95 nm, about 60 nm to about 90 nm, about 60 nm to about 85nm, about 60nm to about 80nm, about 60nm to about 75nm, about 60nm to about 70nm, about 60nm to about 65nm, about 70nm to about 110nm, about 70nm to about 100nm, about 70nm to about 95nm, about 70nm to about 90nm, about 70nm to about 85nm, about 70nm to about 80nm, about 70nm to about 75nm, about 80nm to about 110nm, about 80nm to about 100nm, about 80nm to about 95nm, about 80nm to about 90nm, about 80nm to about 85nm, about 90nm to about 110nm, or about 90nm to about 100nm. According to some embodiments of any of the aspects or embodiments herein, the LNPs are less than about 100 nm in size, e.g., less than about 105 nm, less than about 100 nm, less than about 95 nm, less than about 90 nm, less than about 85 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, less than about 20 nm, less than about 15 nm, or less than about 10 nm in size. According to some embodiments of any of the aspects or embodiments herein, the LNPs are less than about 70 nm in size, e.g., 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, less than about 20 nm, less than about 15 nm, or less than about 10 nm in size. According to some embodiments, the LNPs are less than about 60 nm in size, e.g., 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, less than about 20 nm, less than about 15 nm, or less than about 10 nm in size.

[0018] According to some embodiments of any of the aspects or embodiments herein, the LNP composition has a total lipid to nucleic acid ratio of about 10:1. According to some embodiments of any of the aspects or embodiments herein, the LNP composition has a total lipid to nucleic acid ratio of about 20:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to nucleic acid ratio of about 30:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to nucleic acid ratio of about 40:1. According to some embodiments of any of the aspects or embodiments herein, the composition has a total lipid to nucleic acid ratio of about 50:1.

[0019] According to some embodiments of any of the aspects or embodiments herein, the LNP further comprises a tissue-targeting moiety. The tissue-targeting moiety can be a peptide, oligosaccharide, or the like that can be used to deliver the LNP to one or more specific tissues, such as cancer, liver, CNS, or muscle. According to some embodiments of any of the aspects or embodiments herein, the tissue-targeting moiety is attached to a PEG-lipid conjugate. According to some embodiments of any of the aspects or embodiments herein, the tissue-targeting moiety is a ligand for a liver-specific receptor. According to some embodiments of any of the aspects or embodiments herein, the ligand for the liver-specific receptor used for liver targeting is an oligosaccharide, such as N-acetylgalactosamine (GalNAc).

[0020] According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the lipid nanoparticle at a molar percentage of 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.2%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.3%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.4%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.5%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.6%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.7%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.8%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 0.9%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 1.0%.According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of about 1.5%. According to some embodiments of any of the aspects or embodiments herein, the GalNAc-linked PEG-lipid conjugate is present in the LNP at a molar percentage of 2.0%.

[0021] According to some embodiments of any of the aspects or embodiments herein, the LNP composition is prepared in a buffer, such as malic acid. In some embodiments of any of the aspects or embodiments herein, the composition is prepared at about 10 mM to about 30 mM malic acid, e.g., about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 25 mM, about 15 mM to about 20 mM, or about 20 mM to about 25 mM. According to some embodiments of any of the aspects or embodiments herein, the composition is prepared with about 10 mM malic acid, about 11 mM malic acid, about 12 mM malic acid, about 13 mM malic acid, about 14 mM malic acid, about 15 mM malic acid, about 16 mM malic acid, about 17 mM malic acid, about 18 mM malic acid, about 19 mM malic acid, about 20 mM malic acid, 21 mM malic acid, about 22 mM malic acid, about 23 mM malic acid, about 24 mM malic acid, about 25 mM malic acid, about 26 mM malic acid, about 27 mM malic acid, about 28 mM malic acid, about 29 mM malic acid, or about 30 mM malic acid. According to some embodiments of any of the aspects or embodiments herein, the composition comprises about 20 mM malic acid.

[0022] According to some embodiments of any of the aspects or embodiments herein, the LNP composition is prepared in a solution having about 30 mM to about 50 mM NaCl, e.g., about 30 mM to about 45 mM NaCl, about 30 mM to about 40 mM NaCl, about 30 mM to about 35 mM NaCl, about 35 mM to about 45 mM NaCl, about 35 mM to about 40 mM NaCl, or about 40 mM to about 45 mM NaCl. According to some embodiments of any of the aspects or embodiments herein, the LNP composition is prepared in a solution having about 30 mM NaCl, about 35 mM NaCl, about 40 mM NaCl, or about 45 mM NaCl. According to some embodiments of any of the aspects or embodiments herein, the LNP composition is prepared in a solution having about 40 mM NaCl.

[0023] According to some embodiments of any of the aspects or embodiments herein, the LNP composition comprises about 20 mM to about 100 mM MgCl, e.g., about 20 mM to about 90 mM MgCl, about 20 mM to about 80 mM MgCl, about 20 mM to about 70 mM MgCl, about 20 mM to about 60 mM MgCl, about 20 mM to about 50 mM MgCl, or about 20 mM to about 50 mM MgCl. 0 mM MgCl2, about 20 mM to about 40 mM MgCl2, about 20 mM to about 30 mM MgCl2, about 320 mM to about 90 mM MgCl2, about 30 mM to about 80 mM MgCl2, about 30 mM to about 70 mM MgCl2, about 30 mM to about 60 mM MgCl2, about 30 mM to about 50 mM MgCl2, about 30 mM to about 40 mM MgCl2, about 40 mM to about 90 mM MgCl2, about 40 mM to about 80 mM MgCl2, about 40 mM to about 70 mM MgCl2, about 40 mM to about 60 mM MgCl2, about 40 mM to about 50 mM MgCl2, about 50 mM to about 90 mM MgCl2, about 50 mM to about 80 mM MgCl2, about 50 mM to about 70 mM It is prepared in a solution having about 50 mM to about 60 mM MgCl, about 60 mM to about 90 mM MgCl, about 60 mM to about 80 mM MgCl, about 60 mM to about 70 mM MgCl, about 70 mM to about 90 mM MgCl, about 70 mM to about 80 mM MgCl, or about 80 mM to about 90 mM MgCl.

[0024] According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a closed-ended linear double-stranded DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises an expression cassette comprising a promoter sequence and a transgene.

[0025] According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises an expression cassette comprising a polyadenylation sequence.

[0026] According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises at least one inverted terminal repeat (ITR) flanking either the 5' or 3' end of the expression cassette. According to some embodiments of any of the aspects or embodiments herein, the expression cassette is flanked by two ITRs, the two ITRs comprising one 5' ITR and one 3' ITR. According to some embodiments of any of the aspects or embodiments herein, the expression cassette is linked to the ITR at its 3' end (3' ITR). According to some embodiments of any of the aspects or embodiments herein, the expression cassette is linked to the ITR at its 5' end (5' ITR). According to some embodiments of any of the aspects or embodiments herein, at least one of the 5' ITR and the 3' ITR is a wild-type AAV ITR. According to some embodiments of any of the aspects or embodiments herein, at least one of the 5' ITR and the 3' ITR is a modified ITR. According to some embodiments of any of the aspects or embodiments herein, the ceDNA further comprises a spacer sequence between the 5' ITR and the expression cassette.

[0027] According to some embodiments of any of the aspects or embodiments herein, the ceDNA further comprises a spacer sequence between the 3' ITR and the expression cassette. According to some embodiments of any of the aspects or embodiments herein, the spacer sequence is at least 5 base pairs in length. According to some embodiments of any of the aspects or embodiments herein, the spacer sequence is 5 to 100 base pairs in length. According to some embodiments of any of the aspects or embodiments herein, the spacer sequence is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 base pairs in length. According to some embodiments of any of the aspects or embodiments herein, the spacer sequence is 5 to 500 base pairs in length. According to some embodiments of any of the aspects or embodiments herein, the spacer sequence is 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240 , 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, or 495 base pairs long.

[0028] According to some embodiments of any of the aspects or embodiments herein, the ceDNA has a nick or a gap.

[0029] According to some embodiments of any of the aspects or embodiments herein, the ITRs are derived from an AAV serotype, such as a goose virus ITR, a B19 virus ITR, or a wild-type ITR from a parvovirus. According to some embodiments of any of the aspects or embodiments herein, the AAV serotype is selected from the group including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0030] According to some embodiments of any of the aspects or embodiments herein, the ITR is a mutant ITR, and the ceDNA optionally comprises an additional ITR different from the first ITR. According to some embodiments of any of the aspects or embodiments herein, the ceDNA comprises two mutant ITRs at both the 5' and 3' ends of the expression cassette, and optionally the two mutant ITRs are symmetric mutants. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is CELiD, a DNA-based minicircle, MIDGE, ministering DNA, a dumbbell-shaped linear double-stranded closed-end DNA comprising two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette, or doggybone™ DNA. According to some embodiments of any of the aspects or embodiments herein, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0031] According to some aspects, the present disclosure provides a method of treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition according to any of the aspects or embodiments herein. According to some embodiments of any of the aspects or embodiments herein, the subject is a human. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is selected from the group consisting of 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, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Scheie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II HS), and the like. II), Sanfilippo types A, B, C, and D (MPS III A, B, C, and D), Morquio 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 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, Fabry disease, cystinosis, Batten disease disease, aspartylglucosaminuria, Salla disease, Danon disease (LAMP-2 deficiency), lysosomal acid lipase (LAL) deficiency, neuronal ceroid lipofuscinosis (CLN1-8, INCL, and LINCL), sphingolipidosis, galactosialidosis, amyotrophic lateral sclerosis (ALS), Parkinson's disease, Alzheimer's disease, Huntington's disease, spinocerebellar ataxia, spinal muscular atrophy, Friedreich's ataxia,The genetic disorder is selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), dystrophic epidermolysis bullosa (DEB), ectonucleotide pyrophosphatase 1 deficiency, generalized arterial calcification of infancy (GACI), Leber congenital amaurosis, Stargardt macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, 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 aspects or embodiments herein, the genetic disorder is Leber congenital amaurosis (LCA). According to some embodiments of any of the aspects or embodiments herein, the LCA is LCA10. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Niemann-Pick disease. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Stargardt's macular dystrophy. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disease type I) or Pompe disease (glycogen storage disease type II). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is hemophilia A (factor VIII deficiency). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is hemophilia B (factor IX deficiency). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Hunter syndrome (mucopolysaccharidosis II). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is cystic fibrosis. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is dystrophic epidermolysis bullosa (DEB). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is:According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is phenylketonuria (PKU). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is progressive familial intrahepatic cholestasis (PFIC). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Wilson's disease. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Gaucher disease type I, II, or III. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is age-related macular degeneration. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is ornithine transcarbamylase deficiency. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is retinitis pigmentosa (RP1). According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is Usher syndrome. According to some embodiments of any of the aspects or embodiments herein, the genetic disorder is lysosomal acid lipase (LAL) deficiency. [Brief explanation of the drawings]

[0032] Embodiments of the present disclosure, briefly summarized above and discussed in more detail below, can be understood by reference to exemplary embodiments of the present disclosure, which are depicted in the accompanying drawings. However, the accompanying drawings illustrate only typical embodiments of the present disclosure and therefore should not be considered limiting in scope, as the present disclosure may admit of other equally effective embodiments.

[0033] [Figure 1] 1 shows the improvement in ceDNA-luc expression achieved by using the disclosed lipid nanoparticles (e.g., LNP5 containing lipid 1 and LNP6 containing lipid 3) compared to SS-OPs (e.g., LNP1, 2, and 7-12) observed in Study A. [Figure 2]1 shows the improvement in ceDNA-luc expression achieved by using the disclosed lipid nanoparticles (e.g., LNP16 with lipid 2, LNP17 with lipid 1, and LNP18 with lipid 3) compared to SS-OP (i.e., LNP13) observed in Study B. [Figure 3] The increased doses administered to mice thereby demonstrate improved responsiveness to increased dose levels resulting in a greater increase in ceDNA-luc expression achieved by using the disclosed lipid nanoparticles (e.g., LNP20 with Lipid 1) compared to the SS-OP (i.e., LNP19) observed in Study C. [Figure 4] (Figure 4A) Shows the improvement in ceDNA-luc expression achieved by using the disclosed lipid nanoparticles (e.g., LNP24 with lipid 6, LNP25 with lipid 7, and LNP26 with lipid 8) compared to SS-OP (i.e., LNP23) observed in Study D. (Figure 4B) Shows the improvement in tolerability in mice (as measured by change in body weight) achieved by using the disclosed lipid nanoparticles (e.g., LNP24 with lipid 6, LNP25 with lipid 7, and LNP26 with lipid 8) compared to ionizable lipid A (i.e., LNP22) used as a control. [Figure 5] (FIG. 5A) shows the improvement in ceDNA-luc expression achieved by using the disclosed lipid nanoparticles (e.g., LNP28 containing lipid 9 and LNP29 containing lipid 10) compared to SS-OP (i.e., LNP27). (FIG. 5B) shows that the improvement in ceDNA-luc expression shown in FIG. 5A did not compromise the tolerability of the disclosed lipid nanoparticles in mice. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs), such as viral or non-viral vectors (e.g., closed-end DNA), which can translocate from the cytoplasm to the nucleus of cells and maintain high levels of expression. For example, immunogenicity associated with viral vector-based gene therapy has limited the number of patients that can be treated due to pre-existing background immunity and has prevented patient re-administration to titrate to an effective level for each patient or to maintain efficacy over time. Furthermore, other nucleic acid modalities suffer significantly from immunogenicity due to innate DNA or RNA sensing mechanisms that trigger a cascade of immune responses. Due to the lack of pre-existing immunity, the TNA lipid particles (e.g., lipid nanoparticles) described herein allow for additional doses of TNAs, such as mRNA, siRNA, or ceDNA, as needed, further expanding patient access to include pediatric populations, who may require subsequent administrations depending on tissue growth. Furthermore, it is a discovery of the present disclosure that lipid compositions containing one or more tertiary amino groups and TNA lipid particles (e.g., lipid nanoparticles) containing disulfide bonds, in particular, provide more efficient delivery of TNAs (e.g., ceDNA), better tolerability, and an improved safety profile. Because the TNA lipid particles (e.g., lipid nanoparticles) described herein are free from the packaging constraints imposed by the space within a viral capsid, theoretically the only size limitation of the TNA lipid particles (e.g., lipid nanoparticles) resides in the host cell's expression (e.g., DNA replication or RNA translation) efficiency.

[0035] One of the biggest hurdles in developing therapies, especially for rare diseases, is the large number of individual pathologies. Approximately 350 million people worldwide live with a rare disorder, and the National Institutes of Health defines a rare disorder as a disorder or condition diagnosed in fewer than 200,000 people. Approximately 80% of these rare disorders are genetic in origin, and approximately 95% of them do not have FDA-approved treatments (rarediseases.info.nih.gov / diseases / pages / 31 / faqs-about-rare-diseases). Among the advantages of the TNA lipid particles (e.g., lipid nanoparticles) described herein is that they provide an approach that can be rapidly adapted to multiple diseases that can be treated with specific TNA modalities, particularly rare monogenic diseases, which could meaningfully change the current state of treatment for many genetic disorders or diseases.

[0036] I. Definition The term "alkyl" refers to a univalent saturated, straight-chain (i.e., unbranched) or branched-chain hydrocarbon radical. Exemplary alkyl groups include, but are not limited to, ethyl, propyl, isopropyl, 2-methyl-1-butyl, 3-methyl-2-butyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decanyl, undecanyl, dodecanyl, tridecanyl, tetradecanyl, pentadecanyl, hexadecanyl, heptadecanyl, octadecanyl, nonadecanyl, eicosanyl, and the like.

[0037] The term "alkenyl" refers to a straight-chain or branched aliphatic hydrocarbon radical having one or more (e.g., one or two) carbon-carbon double bonds; alkenyl radicals include radicals having "cis" and "trans" orientations, or, alternatively, "E" and "Z" orientations.

[0038] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of an ionizable lipid of the present invention. 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 include 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 of the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. When 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.

[0039] As used in this specification and the appended claims, the term "about," when referring to a measurable value such as an amount, temporal duration, etc., is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, even more preferably ±0.5%, and even more preferably ±0.1% from the specified value as appropriate for practicing the disclosed methods.

[0040] As used herein, "comprise," "comprising," and "comprises," and "comprised of" are meant to be synonymous with the terms "include," "including," "includes," or "contain," "containing," "contains," e.g., are inclusive or open-ended terms specifying the presence of what follows the components, and do not exclude or preclude the presence of additional, unrecited components, features, elements, members, steps that are known in the art or disclosed therein.

[0041] The term "consisting of" refers to compositions, methods, processes, and their respective components described herein, excluding any elements not recited in the description of the embodiment.

[0042] 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 and novel or functional characteristics of the embodiment of this invention.

[0043] As used herein, the terms "administration," "administering," and variations thereof refer to the introduction of a composition or agent (e.g., a nucleic acid, particularly ceDNA) into a subject, including simultaneous and sequential introduction of one or more compositions or agents. "Administration" can refer, for example, to therapeutic, pharmacokinetic, diagnostic, research, placebo, and experimental methods. "Administration" also encompasses in vitro and ex vivo treatments. Introduction of a composition or agent into a subject is by any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intratumoral, or topical. Administration includes self-administration and administration by another. Administration can be performed by any suitable route. A suitable route of administration allows the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein. In one aspect of any of the aspects or embodiments herein, "administration" refers to therapeutic administration.

[0044] As used herein, phrases such as "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 are meant to refer to any unwanted immune response to a therapeutic nucleic acid, whether viral or non-viral in origin. In some embodiments of any of the aspects or embodiments herein, the unwanted immune response is an antigen-specific immune response to the viral transfer vector itself. In some embodiments of any of the aspects or embodiments herein, the immune response is specific to the transfer vector, which may be double-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.

[0045] As used herein, the terms "carrier" and "excipient" are meant to include 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 pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. 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.

[0046] As used herein, the term "ceDNA" refers to capsid-free, closed-ended, linear, double-stranded (ds) duplex DNA for synthetic or other non-viral gene transfer. A detailed description of ceDNA is provided in International Application 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 Application Nos. PCT / US18 / 49996, filed September 7, 2018, and PCT / US2018 / 064242, filed December 6, 2018, each of which is incorporated herein by reference in its entirety. Certain methods for producing synthetic ceDNA vectors containing various ITR sequences and configurations are described, for example, in International 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. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a closed-end linear double-stranded (CELiD) CELiD DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a DNA-based minicircle. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a minimal immunologically defined gene expression (MIDGE) vector. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is ministering DNA. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is a dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette. According to some embodiments of any of the aspects or embodiments herein, the ceDNA is doggybone™ DNA.

[0047] As used herein, the term "ceDNA-bacmid" is meant to refer to an infectious baculovirus genome that contains a ceDNA genome as an intermolecular duplex that can be propagated as a plasmid in E. coli, thereby acting as a shuttle vector for baculovirus.

[0048] As used herein, the term "ceDNA-baculovirus" is meant to refer to a baculovirus that contains a ceDNA genome as an intermolecular duplex within the baculovirus genome.

[0049] As used herein, the terms "ceDNA-baculovirus-infected insect cells" and "ceDNA-BIIC" are used interchangeably and are meant to refer to invertebrate host cells (including but not limited to insect cells (e.g., Sf9 cells)) infected with a ceDNA-baculovirus.

[0050] As used herein, the term "ceDNA genome" is meant to refer to an expression cassette that further incorporates at least one inverted terminal repeat region. The ceDNA genome may further comprise one or more spacer regions. In some embodiments of any of the aspects and embodiments herein, the ceDNA genome is incorporated into a plasmid or viral genome as an intermolecular double-stranded polynucleotide of DNA.

[0051] As used herein, the terms "DNA regulatory sequence," "control element," and "regulatory element" are used interchangeably herein and are meant to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, proteolysis signals, etc., that provide for 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 the encoded polypeptide.

[0052] As used herein, the term "exogenous" is meant to refer 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 is not normally found and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where 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 is found in relatively low amounts and that has been introduced into a biological system, such as a cell or organism, by a process involving the hand of man, where 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, as used herein, the term "endogenous" refers to a substance that is native to a biological system or cell.

[0053] As used herein, the term "expression" is meant to refer to the cellular processes involved in the production of RNA and proteins, and, where appropriate, secreted proteins, including, for example, but not limited to, transcription, transcription processing, translation, and protein folding, modification, and processing. 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.

[0054] As used herein, the term "expression vector" is meant to refer to a vector that directs the expression of RNA or polypeptides from sequences linked to transcriptional regulatory sequences on the vector. The expressed sequences are often, but not necessarily, heterologous to the host cell. Expression vectors can contain additional elements; for example, an expression vector can have two replication systems, allowing it to be maintained in two organisms, such as human cells for expression and prokaryotic hosts for cloning and amplification. An expression vector may be a recombinant vector.

[0055] As used herein, the terms "expression cassette" and "expression unit" are used interchangeably and are meant to 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 may also be of AAV origin.

[0056] As used herein, the term "flanking" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is flanked on either side 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 the flanked sequence. In one embodiment of any of the aspects or embodiments herein, the term flanking refers to the terminal repeats at each end of a linear, single-stranded synthetic AAV vector.

[0057] As used herein, the term "gene" is used broadly to refer to any segment of nucleic acid involved in the expression of a given RNA or protein in vitro or in vivo. Thus, a gene includes a region that encodes the expressed RNA (usually including a polypeptide-coding sequence) and often the regulatory sequences required for their expression. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and may include sequences specifically designed to have desired parameters.

[0058] As used herein, the phrase "genetic disease" or "genetic disorder" is meant to refer to a disease that is caused, directly or indirectly, in part or in whole, by one or more abnormalities in the genome, particularly conditions that are present from birth. The abnormality can be a mutation, insertion, or deletion in a gene. The abnormality can affect the coding sequence of the gene or its regulatory sequence.

[0059] As used herein, the term "heterologous" is meant to refer to a nucleotide or polypeptide sequence that is not found in a naturally occurring nucleic acid or protein, respectively. A heterologous nucleic acid sequence may 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 may be linked (e.g., by genetic engineering) to a variant polypeptide to generate a nucleotide sequence that encodes a fusion variant polypeptide.

[0060] As used herein, the term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid therapeutic of the present disclosure. By way of non-limiting example, host cells can include isolated primary cells, pluripotent stem cells, CD34 + The host cell may be a cell, an induced pluripotent stem cell, or any of several immortalized cell lines (e.g., HepG2 cells). Alternatively, the host cell may be a cell in situ or in vivo in a tissue, organ, or organism. Furthermore, the host cell may be, for example, a target cell in a mammalian subject (e.g., a human patient in need of gene therapy).

[0061] As used herein, an "inducible promoter" is meant to refer to one that is characterized by initiating or enhancing transcriptional activity when in the presence of, affected by, or contacted by an inducer or inducer agent. As used herein, an "inducer" or "inducer agent" can be endogenous or can be a compound or protein, usually exogenous, administered in such a way that it is active in inducing transcriptional activity from the inducible promoter. In some embodiments of any of the aspects and embodiments herein, the inducer or inducer agent, i.e., a chemical, compound, or protein, can itself be the result of transcription or expression of a nucleic acid sequence (i.e., the inducer can be an inducer protein expressed by another component or module) and can itself be under the control of the inducible promoter. In some embodiments of any of the aspects and embodiments herein, the inducible promoter is induced in the absence of a certain agent, 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.

[0062] As used herein, the term "in vitro" is meant to refer to assays and methods that do not require the presence of cells with intact membranes, such as a cell extract, and can refer to introducing a programmable synthetic biological circuit into a non-cellular system, e.g., a medium that does not contain cells or cell systems, such as a cell extract.

[0063] As used herein, the term "in vivo" is meant to refer to an assay or process that occurs in or within an organism, such as a multicellular animal. In some of the aspects described herein, the method or use may be said to occur "in vivo" when a unicellular organism, such as a bacterium, is used. The term "ex vivo" refers to methods and uses that are carried out using living cells with intact membranes outside the body of 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.

[0064] As used herein, the term "lipid" is meant to refer to a group of organic compounds, including, but not limited to, esters of fatty acids, which are 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 and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0065] 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 lacking phosphorus, such as sphingolipids, glycosphingolipid families, diacylglycerols, and β-acyloxyacids, are also included in the group called amphipathic lipids.In addition, the above amphipathic lipids can be mixed with other lipids, including triglycerides and sterols.

[0066] In one embodiment of any of the aspects or embodiments herein, the lipid composition comprises one or more tertiary amino groups, one or more phenyl ester linkages, and a disulfide linkage.

[0067] As used herein, the term "lipid conjugate" is meant to refer to a conjugated lipid that inhibits aggregation of lipid particles (e.g., lipid nanoparticles). Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as PEG conjugated with dialkyloxypropyl (e.g., PEG-DAA conjugates), PEG conjugated with diacylglycerol (e.g., PEG-DAG conjugates), PEG conjugated with cholesterol, PEG conjugated with phosphatidylethanolamine, and PEG conjugated with ceramide (see, e.g., U.S. Pat. No. 5,885,613), ionized PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates, see, e.g., U.S. Provisional Application Nos. 61 / 294,828, filed January 13, 2010, and 61 / 295,140, ​​filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in PCT Publication WO 2010 / 006282. PEG or POZ can be directly conjugated to the lipid or linked to the lipid via a linker moiety. Any linker moiety suitable for linking PEG or POZ to the lipid can be used, including, for example, non-ester-containing linker moieties and ester-containing linker moieties. In certain preferred embodiments, a non-ester-containing linker moiety, such as an amide or carbamate, is used. The disclosures of each of the above patent documents are incorporated herein by reference in their entirety for all purposes. The lipid conjugates (e.g., PEG-lipids or PEGylated lipids) described herein can be further covalently linked to useful tissue-targeting moieties known in the art (e.g., N-acetylgalactosamine (GalNAc, mono-, di-, tri-, or tetra-sidechain GalNAc)).

[0068] As used herein, the term "lipid-encapsulated" is meant to refer to lipid particles that provide complete encapsulation, partial encapsulation, or both, of an active or therapeutic agent, such as a nucleic acid (e.g., ASO, mRNA, siRNA, ceDNA, viral vector). In preferred embodiments, the nucleic acid is completely encapsulated within the lipid particle (e.g., to form a lipid particle containing the nucleic acid).

[0069] As used herein, the term "lipid particle" or "lipid nanoparticle" refers to a lipid formulation that can be used to deliver a therapeutic agent, such as a nucleic acid therapeutic (TNA), to a desired target site (e.g., a cell, tissue, organ, etc.) (referred to as a "TNA lipid particle," "TNA lipid nanoparticle," or "TNA LNP"). In one embodiment of any of the aspects or embodiments herein, the lipid particles of the present invention are therapeutic nucleic acid-containing lipid particles, which are typically formed from ionizable lipids, non-cationic lipids, and optionally conjugated lipids that prevent particle aggregation. In other preferred embodiments, a therapeutic agent, such as a therapeutic nucleic acid, can be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. In one embodiment of any of the aspects or embodiments herein, the lipid particle comprises a nucleic acid (e.g., ceDNA) and a lipid comprising one or more tertiary amino groups, one or more phenyl ester bonds, and a disulfide bond.

[0070] The lipid particles of the present invention typically have a diameter of about 20 nm to about 120 nm, about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about The average diameter is about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm or about 150 nm.

[0071] As used herein, 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.

[0072] As used herein, the term "ionizable lipid" refers 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 or 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 charged or neutral lipids refers to the nature of the predominant species, and not all lipids need to exist in a charged or neutral form. Generally, ionizable lipids have a pKa of the protonatable group ranging from about 4 to about 7. In some embodiments of any of the aspects and embodiments herein, ionizable lipids may include "cleavable lipids" or "SS-cleavable lipids." Thus, the term "ionizable lipid" as used herein encompasses both the ionized (or charged) and neutral forms of the lipids of the present invention.

[0073] As used herein, the term "neutral lipid" is meant to refer to any lipid species that exists in either an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerol.

[0074] As used herein, the term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0075] As used herein, the term "non-cationic lipid" is meant to refer to any amphipathic lipid, and any other neutral or anionic lipid.

[0076] As used herein, the term "cleavable lipid" or "SS-cleavable lipid" refers to a lipid comprising a disulfide bond cleavable unit. In one embodiment of any of the aspects or embodiments herein, the cleavable lipid comprises a tertiary amine that responds to disulfide bonds, which can be cleaved in an acidic compartment, for example, an endosome or lysosome for membrane destabilization, and in a reducing environment, such as the cytoplasm. In one embodiment of any of the aspects or embodiments herein, the cleavable lipid is an ionizable lipid. In one embodiment of any of the aspects or embodiments herein, the cleavable lipid is a cationic lipid. In one embodiment of any of the aspects or embodiments herein, the cleavable lipid is an ionizable cationic lipid. Cleavable lipids are described in more detail herein.

[0077] As used herein, the term "organic lipid solution" is meant to refer to a composition comprising an organic solvent having, in whole or in part, a lipid.

[0078] As used herein, the term "liposome" refers to lipid molecules assembled in a spherical configuration that encloses an internal aqueous volume separated from an aqueous exterior. Liposomes are vesicles with at least one lipid bilayer. Liposomes are typically used as carriers for drug / therapeutic drug delivery in the context of formulation development. They act by fusing with cell membranes and repositioning their lipid structure to deliver drugs or active pharmaceutical ingredients. Liposome compositions for such delivery are typically composed of phospholipids, particularly compounds with phosphatidylcholine groups, although these compositions may also contain other lipids.

[0079] As used herein, the term "local delivery" refers to the direct delivery of an active agent, such as an interfering RNA (e.g., siRNA), to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site, such as a tumor, or other target site, such as an inflammation site, or into a target organ, such as the liver, heart, pancreas, or kidney.

[0080] As used herein, the term "neDNA" or "nicked ceDNA" is meant to refer to closed-end DNA that has a nick or gap of 2 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).

[0081] 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. The DNA may 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 of these groups. The DNA may be in the form of a minicircle, a plasmid, a bacmid, a minigene, a ministring DNA (a linear, covalently closed DNA vector), a closed-end linear double-stranded DNA (CELiD or ceDNA), doggybone™ DNA, 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 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 otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise specified, 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.

[0082] As used herein, the phrases "nucleic acid therapeutic agent," "therapeutic nucleic acid," and "TNA" are used interchangeably and refer to any modality of treatment that uses nucleic acids 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), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or non-viral DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone™ DNA vector, minimal immunologically defined gene expression (MIDGE)-vector, non-viral ministring DNA vector (linear covalently closed DNA vector), and dumbbell-shaped DNA minimal vector ("dumbbell DNA"). As used herein, the term "TNA LNP" refers to a lipid particle containing at least one TNA, as described above.

[0083] As used herein, a "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group.

[0084] 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 it affects its transcription or expression. A promoter can be said to drive expression, or drive transcription, of the nucleic acid sequence it regulates. The phrases "operably linked," "operably positioned," "operably linked," "under control," and "under transcriptional control" indicate that the promoter is in the correct functional location and / or orientation with respect to a nucleic acid sequence and controls transcription initiation and / or expression of that sequence. As used herein, an "inverted promoter" refers to a promoter in which a nucleic acid sequence is in the reverse orientation, such that what was the coding strand is now the non-coding strand, and vice versa. Inverted promoter sequences can be used in various embodiments to modulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0085] 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. A promoter 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. A promoter 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, one will find a transcription initiation site and protein binding domains involved in the binding of RNA polymerase. Eukaryotic promoters often, but not necessarily, contain "TATA" and "CAT" boxes. A variety of promoters, including inducible promoters, can be used to drive the expression of transgenes in the synthetic AAV vectors disclosed herein. The promoter sequence is bounded at its 3' end by a transcription initiation site and extends upstream (5' orientation) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.

[0086] A promoter may be one naturally associated with a gene or sequence, which may 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 may be referred to as "endogenous." Similarly, in some embodiments of any of the aspects and embodiments herein, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. In some embodiments of any of the aspects and embodiments herein, 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 an operably linked encoded nucleic acid sequence 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, and synthetic promoters or enhancers that are not "naturally occurring," i.e., may contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, promoter sequences can be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR, in connection 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 in its entirety). 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 similarly be used.

[0087] As used herein, "Rep binding site" ("RBS") and "Rep binding element" ("RBE") are used interchangeably and are meant to refer to the binding site of a Rep protein (e.g., AAV Rep78 or AAV Rep68) which, upon binding by the Rep protein, allows the Rep protein to carry out 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 identified in AAV2, 5'-GCGCGCTCGCTCGCTC-3'.

[0088] As used herein, the phrase "recombinant vector" is meant to refer to a vector containing a heterologous nucleic acid sequence or a "transgene" that can be expressed in vivo. It should be understood that the vectors described herein can be combined with other suitable compositions and therapies in some embodiments of any of the aspects and embodiments herein. In some embodiments of any of the aspects and embodiments herein, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining a high copy number of the nucleotide of interest in a subject as extrachromosomal DNA, thereby eliminating the potential effects of chromosomal integration.

[0089] As used herein, the term "reporter" is meant to refer to a protein that can be used to provide a detectable readout. Reporters generally produce 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.

[0090] As used herein, the terms "sense" and "antisense" are meant to refer to the orientation of a structural element on a polynucleotide. The sense and antisense versions of an element are the reverse complements of each other.

[0091] 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 Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably 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.

[0092] As used herein, the term "spacer region" is meant to refer to an intervening sequence that separates functional elements within a vector or genome. In some embodiments of any of the aspects and embodiments herein, the AAV spacer region maintains two functional elements in a desired arrangement for optimal functionality. In some embodiments of any of the aspects and embodiments herein, the spacer region provides or increases the genetic stability of the vector or genome. In some embodiments of any of the aspects and embodiments herein, the spacer region facilitates easy genetic manipulation of the genome by providing a convenient location for cloning sites and gaps of a designed number of base pairs. For example, in certain aspects, an oligonucleotide "polylinker" or "polycloning site" containing several restriction endonuclease sites, or a non-open reading frame sequence designed to lack known protein (e.g., transcription factor) binding sites, can be positioned in the vector or genome to separate cis-acting elements, e.g., inserting a 6mer, 12mer, 18mer, 24mer, 48mer, 86mer, 176mer, etc.

[0093] As used herein, the term "subject" refers to a human or animal to which treatment, including prophylactic treatment, with a therapeutic nucleic acid according to the present invention is provided. Typically, the animal is a vertebrate, such as, but not limited to, a primate, a rodent, a domestic animal, or a game animal. 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 may be male or female. Additionally, the subject may be an infant or child. In some embodiments of any of the aspects and embodiments herein, the subject may be a newborn or fetal subject, e.g., the subject is in utero. Preferably, the subject is a mammal. The mammal may 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 may be advantageously used as subjects representing animal models of diseases and disorders. In addition, the methods and compositions described herein may be used with domestic animals and / or pets. Human subjects may be of any age, sex, race, or ethnic group, e.g., Caucasian (white), Asian, African, Black, African-American, African-European, Latino, Middle Eastern, etc. In some embodiments of any of the aspects and embodiments herein, the subject may be a patient or other subject in a clinical setting. In some embodiments of any of the aspects and embodiments herein, the subject is already undergoing treatment. In some embodiments of any of the aspects and embodiments herein, the subject is an embryo, fetus, newborn, infant, child, adolescent, or adult.In some embodiments of any of the aspects and embodiments herein, the subject is a human fetus, a human newborn, a human infant, a human child, a human adolescent, or a human adult. In some embodiments of any of the aspects and embodiments herein, the subject is an animal embryo, or a non-human embryo or a non-human primate embryo. In some embodiments of any of the aspects and embodiments herein, the subject is a human embryo.

[0094] As used herein, the phrase "subject in need", unless the context and usage of the phrase dictates otherwise, refers to a subject: (i) who is to be administered TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; (ii) who is receiving TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention; or (iii) who has received TNA lipid particles (or a pharmaceutical composition comprising TNA lipid particles) in accordance with the described invention.

[0095] As used herein, the terms "suppress," "reduce," "interfere," "inhibit," and / or "reduce" (and similar terms) generally refer to the act of directly or indirectly decreasing a concentration, level, function, activity, or behavior relative to natural, expected, or average, or relative to a control condition.

[0096] As used herein, the terms "synthetic AAV vector" and "synthetic production of AAV vector" are meant to refer to AAV vectors and methods for their synthetic production in an entirely cell-free environment.

[0097] As used herein, the term "systemic delivery" refers to the delivery of lipid particles that results in widespread biodistribution of an active agent, such as an interfering RNA (e.g., siRNA), within an organism. Some administration techniques result in systemic delivery of a particular agent, while others do not. Systemic delivery means that a useful, preferably therapeutic, amount of the agent is exposed to most parts of the body. Widespread biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or excreted (by first-pass organs (liver, lung, etc.) or by rapid nonspecific cellular binding) before reaching disease sites distal to the administration site. Systemic delivery of lipid particles (e.g., lipid nanoparticles) can be by any means known in the art, including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles (e.g., lipid nanoparticles) is by intravenous delivery.

[0098] As used herein, the terms "terminal resolution site" and "TRS" are used interchangeably herein and are meant to refer to the region where Rep forms a tyrosine-phosphodiester bond with the 5' thymidine that generates 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 can participate in a coordinate ligation reaction.

[0099] As used herein, the terms "therapeutic amount," "therapeutically effective amount," "effective amount," "effective amount," or "pharmaceutically effective amount" of an active agent (e.g., a TNA lipid particle described herein) are used interchangeably to refer to an amount sufficient to provide the intended benefit or effect of treatment, e.g., inhibition of expression of a target sequence compared to the expression level detected in the absence of a therapeutic nucleic acid. Suitable assays for measuring expression of a target gene or target sequence include, for example, examination of protein or RNA levels using techniques known to those skilled in the art, such as dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, and phenotypic assays also known to those skilled in the art. Dosage levels are based on a variety of factors, including the type of injury, age, weight, sex, patient condition, severity of the condition, route of administration, and the specific active agent used. Thus, dosing regimens can vary widely but can be routinely determined by a physician using standard methods. Additionally, the terms "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" include prophylactic or preventative amounts of the described compositions of the present invention. In prophylactic or preventative uses of the described invention, a pharmaceutical composition or medicament is administered to a patient susceptible to or otherwise at risk of a disease, disorder, or condition, including the biochemical, histological, and / or behavioral symptoms of the disease, disorder, or condition, its complications, and intermediate pathological phenotypes manifested during the development of the disease, disorder, or condition, in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay the onset of the disease, disorder, or condition. It is generally preferred to use the maximum dose, i.e., the highest safe dose, according to some medical judgment. The terms "dose" and "administration" are used interchangeably herein. In one aspect of any of the aspects or embodiments herein, "therapeutic amount," "therapeutically effective amount," and "pharmaceutically effective amount" refer to non-prophylactic or non-preventative uses.

[0100] As used herein, the term "therapeutic effect" refers to a result of treatment, which result is deemed to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the prevention, reduction, or elimination of disease symptoms. A therapeutic effect can also include, directly or indirectly, the prevention, reduction, or elimination of the progression of disease symptoms.

[0101] For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from preliminary in vitro studies and / or animal models. The therapeutically effective dose can also be determined from human data. The applied dose can be adjusted based on the relative bioavailability and efficacy of the administered compound. Adjusting the dose to achieve maximum efficacy based on the above methods and other well-known methods is within the ability of a person skilled in the art. The following summarizes general principles for determining therapeutic efficacy, which can be found in Chapter 1 of Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference.

[0102] Pharmacokinetic principles provide the basis for modifying dosing regimens to achieve the desired degree of therapeutic effect while minimizing unacceptable side effects. In situations where the plasma concentration of a drug can be measured and is related to the therapeutic window, additional guidance regarding dosage modifications is available.

[0103] As used herein, the terms "treat," "treating," and / or "treatment" include suppressing, inhibiting, slowing, or reversing the progression of a condition, ameliorating clinical symptoms of a condition, or preventing the appearance of clinical symptoms of a condition, or achieving beneficial or desired clinical results. 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 were previously asymptomatic for the disorder. In one aspect of any of the aspects or embodiments herein, the terms "treat," "treating," and / or "treatment" include suppressing, inhibiting, slowing, or reversing the progression of a condition, or ameliorating clinical symptoms of a condition.

[0104] Beneficial or desired clinical results, such as 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 symptoms of 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.

[0105] The term "vector" or "expression vector" is meant to refer to 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 to bring about the 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 a gene sequence to a cell. In some embodiments of any of the aspects and embodiments herein, the vector can be a recombinant vector or an expression vector.

[0106] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Members of each group may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to include the modified group, thus satisfying all Markush group descriptions used in the appended claims.

[0107] In some embodiments of any of the aspects, the disclosure described herein does not pertain to human cloning processes, processes for correcting the genetic identity of human germ lines, the use of human embryos for industrial or commercial purposes, or animals that are likely to cause suffering without providing any substantial medical benefit to humans or animals, and processes for correcting the genetic identity of animals resulting from such processes.

[0108] Other terms are defined herein within the description of various aspects of the invention.

[0109] All patents and other publications cited throughout this application, including literature references, issued patents, published patent applications, and pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that 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 any admission as to the accuracy of the dates or contents of these documents.

[0110] 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 specific embodiments and examples of the present disclosure have been 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 may be presented in a given order, alternative embodiments may perform the functions in a different order, or the functions may be performed substantially simultaneously. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the present disclosure can be modified, as appropriate, to employ the compositions, functions, and concepts of the above-mentioned references and applications to provide further embodiments of the present disclosure. Furthermore, due to considerations of biological functional equivalence, some changes can be made to protein structure without affecting the type or amount of biological or chemical activity. 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.

[0111] Particular 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 have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments necessarily exhibit such advantages, to be within the scope of the present disclosure.

[0112] The techniques described herein are further illustrated by the following examples, which should not be construed as further limiting in any way. It is understood that the present invention is not limited in any way to the particular methodology, protocols, and reagents, etc., described herein and 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 present invention, which is defined solely by the claims.

[0113] II. Lipids In a first chemical embodiment, an ionizable lipid of formula (I) is provided, [ka] During the ceremony, 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.

[0114] In a second chemical embodiment, the ionizable lipid of formula (I) is that of formula (II): [ka] or a pharmaceutically acceptable salt thereof, 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 remainder of the variables are as described for formula (I).

[0115] In a third chemical embodiment, c and d in the ionizable lipid of formula (I) or (II), or a pharmaceutically acceptable salt thereof, 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 (I) or (II).

[0116] In a fourth chemical embodiment, c in the ionizable lipid of formula (I) or (II) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (I) or the second or third chemical embodiment. Alternatively, as part of the fourth chemical embodiment, c and d in the ionizable lipid of formula (I) or (II), or a pharmaceutically acceptable salt thereof, are each independently 1, 3, 5, or 7, and the remaining variables are as described for formula (I) or the second or third chemical embodiment.

[0117] In a fifth chemical embodiment, d in the ionizable lipid of formula (I) or (II) is 2, 3, 4, 5, 6, 7, or 8, and the remaining variables are as described for formula (I) or the second or third chemical embodiment. Alternatively, as part of the fifth chemical embodiment, at least one of c and d in the ionizable lipid of formula (I) or (II), or a pharmaceutically acceptable salt thereof, is 7, and the remaining variables are as described for formula (I) or the second, third, or fourth chemical embodiment.

[0118] In a sixth chemical embodiment, the ionizable lipid of formula (I) is of formula (III): [ka] or a pharmaceutically acceptable salt thereof, wherein the remaining variables are as described for formula (I).

[0119] In a seventh chemical embodiment, b in the ionizable lipid of Formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is an integer ranging from 3 to 9, and the remaining variables are as described for Formula (I), or the second, third, fourth, or fifth chemical embodiment. Alternatively, as part of the seventh chemical embodiment, b in the ionizable lipid of Formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 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 (I), or the second, third, fourth, or fifth chemical embodiment. In another alternative, as part of a seventh chemical embodiment, b in the ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is 3, 4, 5, 6, 7, 8, or 9, and the remaining variables are as described for formula (I), or the second, third, fourth, or fifth chemical embodiment.

[0120] In an eighth chemical embodiment, a in the ionizable lipid of Formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is an integer ranging from 2 to 18, and the remaining variables are as described for Formula (I), or the second, third, fourth, fifth, or seventh chemical embodiment. Alternatively, as part of the eighth embodiment, a in the ionizable lipid of Formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is an integer ranging from 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, 3-9, 3-8, 3-7, 3-6, 3-5, 4-18, 4-17, 4-16, 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 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, 126, 127, 128, 131, 132, 133, 134, 135, 141, 142, 143, 144, 145, 146, 1518, 152In another alternative, as part of the eighth embodiment, a in the ionizable lipid of Formula (I), (II), or (III) 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 (I), or the second, third, fourth, fifth, or seventh chemical embodiment.

[0121] In a ninth chemical embodiment, R in an ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 1 is absent or (C5~C 15 ) alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, wherein the remaining variables are as described for formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of a ninth chemical embodiment, R in the ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is 1 is absent or (C5~C 15 ) alkenyl, -C(O)O(C4-C 16 ) alkyl, and (C4-C 16 ) cyclopropyl substituted with alkyl, wherein the remaining variables are as described for formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. Alternatively, as part of a ninth chemical embodiment, R in the ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, is 1 is absent or (C5~C 12 ) alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12) cyclopropyl substituted with alkyl, wherein the remaining variables are as described for formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. In another alternative, as part of a ninth chemical embodiment, R in the ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 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, wherein the remainder of the variables are as described for formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment.

[0122] In a tenth chemical embodiment, R 1 is C 10 alkenyl, wherein the remainder of the variables are as described in any one of the previous embodiments.

[0123] In an eleventh chemical embodiment, R in an ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 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 The alkyl in alkyl is an unbranched alkyl, and the remainder of the variables are as described in any one of the preceding embodiments. In one chemical embodiment, R 1 is —C(O)O(C alkyl). Alternatively, in an eleventh chemical embodiment, R in the ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 1 -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10The alkyl in alkyl is a branched alkyl, and the remainder of the variables are as described in any one of the preceding chemical embodiments. In one chemical embodiment, R 1 is -C(O)O(C 17 alkyl), where the remainder of the variables are as described in any one of the preceding chemical embodiments.

[0124] In a twelfth chemical embodiment, R in an ionizable lipid of formula (I), (II), or (III), or a pharmaceutically acceptable salt thereof, 1 is selected from any of the groups listed in Table 1 below, where the wavy bond in each of the groups indicates the point of attachment of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiment. The present disclosure further provides a method for preparing a lipid comprising administering to a subject a lipid molecule ... 1 Any one of the groups and R in Table 2 2 Combinations with any one of the groups are contemplated, with the remaining variables being as described for Formula (I), or the second, third, fourth, fifth, seventh, or eighth chemical embodiments. [Table 1]

[0125] In a thirteenth chemical embodiment, R in the ionizable lipid of formula (I), or a pharmaceutically acceptable salt thereof, 2 is selected from any of the groups listed in Table 2 below, where the wavy bond in each of the groups indicates the point of attachment of the group to the remainder of the lipid molecule, and the remaining variables are as described for formula (I), or the seventh, eighth, ninth, tenth, or eleventh chemical embodiment. [Table 2]

[0126] Specific examples are provided in the Exemplification section of Table 3 below and are included herein as part of the fourteenth chemical embodiment of the ionizable lipid of Formula (I). Pharmaceutically acceptable salts and ionized and neutral forms are also included. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0127] In a further embodiment, a lipid of formula (Ia), (Ib), or (Ic): [ka] or a pharmaceutically acceptable salt thereof is contemplated herein, wherein R q and R z are each independently an aliphatic group (including alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl) or an aryl group, and the remaining variables are as described above in any one of the preceding chemical embodiments. q and R zare each independently hydrogen or C1-C6 alkyl, and the remaining variables are as described above in any one of the preceding chemical embodiments. The disclosed LNPs, compositions, methods of use, etc. also apply to lipids of formula (Ia), (Ib), or (Ic). Lipids of formula (Ia), (Ib), or (Ic), such as lipids of formula (I), can be prepared by treatment with chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl).

[0128] Additionally, lipids of formula (II) or (III), or any of the exemplary lipids disclosed herein, can be converted to the corresponding quaternary lipids (all contemplated in this disclosure), e.g., lipids of formula (I), by treatment with chloromethane (CHCl) in acetonitrile (CHCN) and chloroform (CHCl).

[0129] Lipid nanoparticles (LNPs) comprising the ionizable lipids described herein and a capsid-free, non-viral vector (e.g., ceDNA), or pharmaceutical compositions thereof, can be used to deliver the capsid-free, non-viral DNA vector to a desired target site (e.g., a cell, tissue, organ, etc.).

[0130] In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation is prepared and loaded with TNA. In one embodiment, the lipid particle (lipid nanoparticle) formulation is prepared and loaded with ceDNA obtained by the process disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein by reference in its entirety. This can be achieved by high-energy mixing of ethanolic lipids with aqueous TNA, such as ceDNA, at low pH, which protonates the lipids and provides favorable energetics for ceDNA / lipid association and nucleation of the particles. The particles can be further stabilized by aqueous dilution and removal of the organic solvent. The particles can be concentrated to a desired level.

[0131] Typically, lipid particles (e.g., lipid nanoparticles) are prepared at a total lipid to nucleic acid (mass or weight) ratio of about 10:1 to 60:1. In some embodiments of any of the aspects and embodiments herein, the lipid to nucleic acid ratio (mass / mass ratio, w / w ratio) can be within the range of about 1:1 to about 60:1, about 1:1 to about 55:1, about 1:1 to about 50:1, about 1:1 to about 45:1, about 1:1 to about 40:1, about 1:1 to about 35:1, about 1:1 to about 30:1, about 1:1 to about 25:1, about 10:1 to about 14:1, about 3:1 to about 15:1, about 4:1 to about 10:1, about 5:1 to about 9:1, about 6:1 to about 9:1, or about 30:1 to about 60:1. According to some embodiments of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) are prepared with a nucleic acid (mass or weight) to total lipid ratio of about 60:1. According to some embodiments of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) are prepared with a nucleic acid (mass or weight) to total lipid ratio of about 30:1. The amounts of lipid and nucleic acid can be adjusted to provide a desired N / P ratio, for example, an N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. Generally, the total lipid content of the lipid particle formulation can range from about 5 mg / mL to about 30 mg / mL.

[0132] In some embodiments of any of the aspects and embodiments herein, the lipid nanoparticles comprise an agent for condensing and / or encapsulating nucleic acid cargo, such as ceDNA. Such agents are also referred to herein as condensing agents or encapsulating agents. Without limitation, any compound known in the art for condensing and / or encapsulating nucleic acids can be used as long as it is non-fusogenic. In other words, the agent can condense and / or encapsulate nucleic acid cargo, such as ceDNA, but has little or no fusion activity. Without wishing to be bound by theory, the condensing agent may have some fusion activity when it does not condense / encapsulate nucleic acid, such as ceDNA, but the nucleic acid encapsulated in the lipid nanoparticles formed with the condensing agent may be non-fusogenic.

[0133] Generally, ionizable lipids are used to condense nucleic acid cargoes, such as ceDNA, at low pH and to induce membrane association and membrane fusion.Generally, cationic lipids are lipids that are positively charged or contain at least one amino group that is protonated under acidic conditions, such as pH 6.5 or less.Cationic lipids can also be ionizable lipids, for example, ionizable cationic lipids."Non-fusogenic ionizable lipid" refers to the ionizable lipid that can condense and / or encapsulate nucleic acid cargoes, such as ceDNA, but has no or little fusogenic activity.

[0134] In one embodiment of any of the aspects or embodiments herein, the ionizable lipids may comprise 20 to 90% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). For example, the ionizable lipid molar content may be 20 to 70% (mol), 30 to 60% (mol), 40 to 60% (mol), 40 to 55% (mol), or 45 to 55% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the ionizable lipids comprise about 50 mol% to about 90 mol% of the total lipids present in the lipid particle (e.g., lipid nanoparticle).

[0135] In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) can further comprise a non-cationic lipid. The non-cationic lipid can help to increase fusogenicity and enhance the stability of the LNP during formation. Non-cationic lipids include amphipathic lipids, neutral lipids, and anionic lipids. Thus, the non-cationic lipid can be a neutral, uncharged, zwitterionic, or anionic lipid. The non-cationic lipid is typically used to enhance membrane fusogenicity.

[0136] Exemplary non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, 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 (DSP), E), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (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 oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,These lipids include, but are not limited to, 2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicaside, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl group in these lipids is preferably C, 10 ~C 24 Acyl groups derived from fatty acids having carbon chains, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0137] Other examples of non-cationic lipids suitable for use in lipid particles (e.g., lipid nanoparticles) include non-phosphorous lipids such as, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0138] In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid is a phospholipid. In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some embodiments of any of the aspects and embodiments herein, the non-cationic lipid is DSPC. In other embodiments, the non-cationic lipid is DOPC. In other embodiments, the non-cationic lipid is DOPE.

[0139] In some embodiments of any of the aspects and embodiments herein, the non-cationic lipids may account for 0 to about 20% (mol) of the total lipids present in the lipid nanoparticles. In some embodiments of any of the aspects and embodiments herein, the non-cationic lipid content is 0.5 to 15% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments herein, the non-cationic lipid content is 5 to 12% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In some embodiments of any of the aspects and embodiments herein, the non-cationic lipid content is 5 to 10% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 6% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 7.0% (mol) of the total lipids present in the lipid particles (e.g., lipid nanoparticles). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 7.5% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 8.0% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 9.0% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the non-cationic lipid content is about 10% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the non-cationic lipid content is about 11% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle).

[0140] Exemplary non-cationic lipids are described in PCT Publication No. WO 2017 / 099823 and U.S. Patent Publication No. 2018 / 0028664, the contents of both of which are incorporated herein by reference in their entireties.

[0141] In some embodiments of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) can further comprise a component such as a sterol to provide membrane integrity and stability to the lipid particles. In some embodiments of any of the aspects or embodiments herein, an exemplary sterol that can be used in the lipid particles is cholesterol or a derivative thereof. 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 of any of the aspects and embodiments herein, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether. In some embodiments of any of the aspects and embodiments herein, the cholesterol derivative is cholestryl hemisuccinate (CHEMS).

[0142] Exemplary cholesterol derivatives are described in PCT International Publication No. WO2009 / 127060 and US Patent Publication No. US2010 / 0130588, the contents of both of which are incorporated herein by reference in their entireties.

[0143] In one embodiment of any of the aspects or embodiments herein, components that provide membrane integrity, such as sterols, may comprise 0-50% (mol) of the total lipids present in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, such components comprise 20-50% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, such components comprise 30-40% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, such components comprise 35-45% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, such components comprise 38-42% (mol) of the total lipid content of the lipid particle (e.g., lipid nanoparticle).

[0144] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) may further comprise polyethylene glycol (PEG) or conjugated lipid molecules. These are generally used to inhibit aggregation and / or provide steric stabilization of the lipid particles (e.g., lipid nanoparticles). Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments of any of the aspects and embodiments herein, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., a (methoxypolyethylene glycol)-conjugated lipid. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, e.g., a PEG 2000 -DMG (dimyristoylglycerol).

[0145] Exemplary PEG-lipid conjugates include PEG-diacylglycerol (DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEGylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEGS-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbamate, N-(carbonyl-methacrylate)-2-(2-methyl-2-propanol), N-( ... Examples of PEG-lipid conjugates include, but are not limited to, hydroxypolyethylene glycol (2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or mixtures thereof. Additional exemplary PEG-lipid conjugates are described in, for example, US 5,885,613, US 6,287,591, US 2003 / 0077829, US 2003 / 0077829, US 2005 / 0175682, US 2008 / 0020058, US 2011 / 0117125, US 2010 / 0130588, US 2016 / 0376224, and US 2017 / 0119904, the contents of all of which are incorporated herein by reference in their entirety.

[0146] In one embodiment of any of the aspects or embodiments herein, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG-dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be, for example, PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG-disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxaotanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-diester), or the like. PEG lipids may be one or more of PEG-DMG, ...

[0147] In one embodiment of any of the aspects or embodiments herein, lipids conjugated with molecules other than PEG can be used instead of PEG-lipids. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic polymer lipid (CPL) conjugates can be used instead of or in addition to PEG-lipids. Exemplary conjugated lipids, i.e., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates, and cationic polymer-lipids, are described in PCT International Patent Application Publication Nos. 1996 / 010392, 1998 / 051278, 2002 / 087541, 2005 / 026372, 2008 / 147438, and 2009 / 102021. Nos. 9 / 086558, 2012 / 000104, 2017 / 117528, 2017 / 099823, 2015 / 199952, 2017 / 004143, 2015 / 095346, 2012 / 000104, 2012 / 000104, and 2010 / 006282, U.S. Patent Nos. Patent application publication numbers 2003 / 0077829, 2005 / 0175682, 2008 / 0020058, 2011 / 0117125, 2013 / 0303587, 2018 / 0028664, 2015 / 0376115, 2016 / 0376224, 2016 / 0317458, Nos. 2013 / 0303587, 2013 / 0303587, and 2011 / 0123453, and U.S. Patent Nos. 5,885,613, 6,287,591, 6,320,017, and 6,586,559, the contents of all of which are incorporated herein by reference in their entireties.

[0148] In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate is present in the lipid nanoparticle at a molar ratio of about 0% to about 20%. In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is 0.5 to 10% (mol) in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is 1 to 5% (mol) in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is 1 to 3% (mol) in the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the PEG-lipid conjugate content is about 1.5% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is about 2% (mol) in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is about 2.5% (mol) in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is about 3% (mol) of the total lipid present in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is about 3% (mol) in the lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the PEG-lipid conjugate content is about 3.5% (mol) in the lipid particle (e.g., lipid nanoparticle).

[0149] In some embodiments of any of the aspects and embodiments herein, the conjugated lipid, such as a PEG-lipid conjugate or a PEGylated lipid, is present at a molar percentage of greater than about 2.0% of the total lipid in the lipid nanoparticle, e.g., about 2.1%, or 2.2%, or 2.3%, or 2.4%, or about 2.5% to about 10%; or about 2.1%, or 2.2%, or 2.3%, or 2.4%, or about 2.5% to about 7.5%; about 2.1%, or 2.2%, or 2.3%, or 2.4%, or about 2.5% to about 5%; about 3% to about 5%; about 3% to about 4.5%; about 3% to about 4%; about 3.5% to about 5%; about 3.5% to about 4.5%, about 2.5% to about 4%; about 2.5% to about 3.5%, or about 2.5% to about 3%.

[0150] It is understood that the molar ratios of the disclosed ionizable lipids to non-cationic lipids, sterols, and PEG-conjugated lipids can be varied as needed. For example, lipid particles (e.g., lipid nanoparticles) can comprise 30-70% lipid by molar or total weight of the composition, 0-60% cholesterol by molar or total weight of the composition, 0-30% non-cationic lipid by molar or total weight of the composition, and 1-10% PEG-conjugated lipid by molar or total weight of the composition. In one embodiment of any of the aspects or embodiments herein, the composition comprises 40-60% ionizable lipid by molar or total weight of the composition, 30-50% cholesterol by molar or total weight of the composition, 5-15% non-cationic lipid by molar or total weight of the composition, and 1-5% PEG-conjugated lipid by molar or total weight of the composition. In one embodiment of any of the aspects or embodiments herein, the composition is 40-60% by molar or total weight of ionizable lipid, 30-40% by molar or total weight of cholesterol, 5-10% by molar or total weight of non-cationic lipid, and 1-5% by molar or total weight of PEG-conjugated lipid. The composition may contain 60-70% by molar or total weight of ionizable lipid, 25-35% by molar or total weight of cholesterol, 5-10% by molar or total weight of non-cationic lipid, and 0-5% by molar or total weight of PEG-conjugated lipid. The composition may also contain up to 45-55% ionizable lipid by molar or total weight of the composition, 35-45% cholesterol by molar or total weight of the composition, 2-15% non-cationic lipid by molar or total weight of the composition, and 1-5% PEG-conjugated lipid by molar or total weight of the composition.Formulations may also be prepared, for example, from 8 to 30% by molar or total weight of the composition of an ionizable lipid, from 5 to 15% by molar or total weight of the composition of a non-cationic lipid, and from 0 to 40% by molar or total weight of the composition of cholesterol; from 4 to 25% by molar or total weight of the composition of an ionizable lipid, from 4 to 25% by molar or total weight of the composition of a non-cationic lipid, from 2 to 25% by molar or total weight of the composition of cholesterol, from 10 to 35% by molar or total weight of the composition of a conjugated lipid, and from 5% by molar or total weight of the composition of cholesterol; or The lipid nanoparticle formulation may comprise 2-30% by molar or total weight of ionizable lipid, 2-30% by molar or total weight of non-cationic lipid, 1-15% by molar or total weight of cholesterol, 2-35% by molar or total weight of PEG-conjugated lipid, and 1-20% by molar or total weight of cholesterol; or even up to 90% by molar or total weight of ionizable lipid and 2-10% by molar or total weight of non-cationic lipid; or even 100% by molar or total weight of ionizable lipid. In some embodiments of any of the aspects and embodiments herein, the lipid particle formulation comprises ionizable lipid, non-cationic phospholipid, cholesterol, and PEGylated lipid (conjugated lipid) in a molar ratio of 50:10:38.5:1.5. In some embodiments of any of the aspects and embodiments herein, the lipid particle formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of 50:10:38:2. In some embodiments of any of the aspects and embodiments herein, the lipid particle formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of 50:10:37:3. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of about 50:7:40:3.In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of about 50:8:40:2. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of about 50:9:39:2. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation comprises an ionizable lipid, a non-cationic phospholipid, cholesterol, and a PEGylated lipid (conjugated lipid) in a molar ratio of about 50:9:38:3.

[0151] In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) comprises an ionizable lipid, a non-cationic lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), and a PEGylated lipid (conjugated lipid), wherein the molar ratio of the lipids is in the range of 20-70 mole percent for the ionizable lipid (target 30-60), the mole percent of the non-cationic lipid is in the range of 0-30 (target 0-15), the mole percent of the sterol is in the range of 20-70 (target 30-50), and the mole percent of the PEGylated lipid (conjugated lipid) is in the range of 1-6 (target 2-5).

[0152] Lipid nanoparticles (LNPs) containing ceDNA are disclosed in International Application No. PCT / US2018 / 050042, filed September 7, 2018, which is incorporated herein in its entirety and are contemplated for use in the methods and compositions disclosed herein.

[0153] The particle size of the lipid particles (e.g., lipid nanoparticles) can be determined by quasi-elastic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) and is approximately 50-150 nm diameter, approximately 55-95 nm diameter, or approximately 70-90 nm diameter.

[0154] The pKa of the formulated ionizable lipid can correlate with the effectiveness of the LNP for delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010), both of which are incorporated herein by reference in their entireties). In one embodiment of any of the aspects or embodiments herein, the pKa of each ionizable lipid is determined in the lipid nanoparticle using a fluorescence-based assay of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Lipid nanoparticles consisting of ionizable lipid / DSPC / cholesterol / PEG-lipid (50 / 10 / 38.5 / 1.5 mol%) in PBS at a concentration of 0.4 mM total lipid can be prepared using the in-line process described herein and elsewhere. TNS can be prepared as a 100 mM stock solution in distilled water. Vesicles can be diluted to 24 mM lipid in 2 mL of buffer solution containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, with a pH ranging from 2.5 to 11. An aliquot of TNS solution can be added to a final concentration of 1 mM, followed by vortex mixing. Emission intensity is measured at room temperature in an SLM Aminco Series 2 luminescence spectrophotometer using an excitation wavelength of 321 nm and an emission wavelength of 445 nm. A sigmoidal best-fit analysis can be applied to the fluorescence data, and the pKa is determined as the pH that produces half-optimal fluorescence intensity.

[0155] In one embodiment of any of the aspects or embodiments herein, relative activity can be determined by measuring luciferase expression in the liver 4 hours after administration via tail vein injection. Activity is compared at doses of 0.3 and 1.0 mg ceDNA / kg and is expressed as ng luciferase / g liver measured 4 hours after administration.

[0156] Without limitation, the lipid particles (e.g., lipid nanoparticles) of the present disclosure include lipid formulations that can be used to deliver capsid-free, non-viral DNA vectors to a desired target site (e.g., a cell, tissue, organ, etc.). Generally, the lipid particles (e.g., lipid nanoparticles) comprise a capsid-free, non-viral DNA vector and an ionizable lipid, or a salt thereof.

[0157] In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) comprises an ionizable lipid / non-cationic lipid / sterol / conjugated lipid in a molar ratio of 50:10:38.5:1.5.

[0158] In one embodiment of any of the aspects or embodiments herein, the present disclosure provides a lipid particle (e.g., lipid nanoparticle) formulation comprising a phospholipid, a lecithin, a phosphatidylcholine, and a phosphatidylethanolamine.

[0159] III. Therapeutic Nucleic Acids (TNA) The present disclosure provides a lipid-based platform for delivering therapeutic nucleic acids (TNAs). Non-limiting examples of RNA-based therapeutics include mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). Non-limiting examples of DNA-based therapeutics include minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or nonviral DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE) vectors, nonviral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). Thus, aspects of the present disclosure generally provide ionizable lipid particles (e.g., lipid nanoparticles) containing TNAs.

[0160] therapeutic nucleic acids Exemplary therapeutic nucleic acids of the present disclosure can include, but are not limited to, minigenes, plasmids, minicircles, small interfering RNAs (siRNAs), microRNAs (miRNAs), antisense oligonucleotides (ASOs), ribozymes, closed-ended double-stranded DNA (e.g., ceDNA, CELiDs, linear covalently closed DNA ("ministring"), doggybone™, protelomeric closed-ended DNA, or dumbbell linear DNA), Dicer substrate dsRNA, small hairpin RNAs (shRNAs), asymmetric interfering RNAs (aiRNAs), microRNAs (miRNAs), mRNAs, tRNAs, rRNAs, and DNA viral vectors, viral RNA vectors, and any combination thereof.

[0161] siRNA or miRNA, which can downregulate the intracellular level of a specific protein through a process called RNA interference (RNAi), are also contemplated as nucleic acid therapeutics by the present invention. After siRNA or miRNA is introduced into the cytoplasm of a host cell, these double-stranded RNA constructs can bind to a protein called RISC. The sense strand of siRNA or miRNA is removed by the RISC complex. When the RISC complex binds to complementary mRNA, it cleaves the mRNA and releases the cleaved strand. RNAi works by inducing specific destruction of mRNA, which leads to the downregulation of the corresponding protein.

[0162] Antisense oligonucleotides (ASOs) and ribozymes, which inhibit mRNA translation into proteins, are potential nucleic acid therapeutics. In the case of antisense constructs, these single-stranded deoxyribonucleotides have sequences complementary to the target protein mRNA and can bind to the mRNA through Watson-Crick base pairing. This binding prevents translation of the target mRNA and / or induces RNase H degradation of the mRNA transcript. As a result, antisense oligonucleotides have enhanced specificity of action (i.e., downregulation of specific disease-related proteins).

[0163] In any of the methods and compositions provided herein, the therapeutic nucleic acid (TNA) can be a therapeutic RNA. The therapeutic RNA can be an inhibitor of mRNA translation, an RNA interference (RNAi) agent, a catalytically active RNA molecule (ribozyme), a transfer RNA (tRNA), or an RNA that binds to an mRNA transcript (ASO), a protein, or other molecular ligand (aptamer). In any of the methods provided herein, the RNAi agent can be double-stranded RNA, single-stranded RNA, microRNA, short interfering RNA, small hairpin RNA, or triple-helix-forming oligonucleotide.

[0164] In any of the method compositions provided herein, the therapeutic nucleic acid (TNA) can be a therapeutic DNA such as a closed-end double-stranded DNA (e.g., ceDNA, CELiD, linear covalently closed DNA ("ministring"), doggybone™, protelomeric closed-end DNA, dumbbell linear DNA, plasmid, minicircle, etc.). Some embodiments of the present disclosure are based on methods and compositions comprising a closed-end linear duplex (ceDNA) capable of expressing a transgene (e.g., a therapeutic nucleic acid). ceDNA vectors as described herein do not have the packaging constraints imposed by the limited space within a viral capsid. ceDNA vectors represent a versatile eukaryotically produced alternative to prokaryotically produced plasmid DNA vectors.

[0165] The ceDNA vector preferably has a linear, continuous structure rather than a discontinuous structure. A linear, continuous structure is believed to be more stable against attack by cellular endonucleases and less likely to undergo recombination, leading to mutagenesis. Therefore, a ceDNA vector with a linear, continuous structure is a preferred embodiment. A continuous, linear, single-stranded, intramolecularly double-stranded ceDNA vector may have covalently linked termini without sequences encoding AAV capsid proteins. These ceDNA vectors are structurally different from plasmids (including the ceDNA plasmids described herein), which are circular, double-stranded nucleic acid molecules of bacterial origin. While the complementary strands of a plasmid can be separated following denaturation to produce two nucleic acid molecules, a ceDNA vector, although having complementary strands, is a single DNA molecule and therefore remains a single molecule even when denatured. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector, unlike a plasmid, can be produced without prokaryotic cell-type DNA base methylation. Thus, ceDNA vectors and ceDNA-plasmids differ both in terms of structure (in particular, linear versus circular) and the methods used to produce and purify these different entities, and also in terms of their DNA methylation, which is of prokaryotic cell type in the case of ceDNA-plasmids and eukaryotic cell type in the case of ceDNA-vectors.

[0166] Provided herein are non-viral capsid-free ceDNA molecules (ceDNA) with covalently closed ends. These non-viral capsid-free ceDNA molecules can be produced in permissive host cells from expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses, or integrating cell lines) containing a heterologous gene (e.g., a transgene, particularly a therapeutic transgene) positioned between two different inverted terminal repeat (ITR) sequences, where the ITRs are different from each other. In some embodiments of any of the aspects and embodiments herein, one of the ITRs is modified by deletion, insertion, and / or substitution compared to the wild-type ITR sequence (e.g., AAV ITR), and at least one of the ITRs contains a functional terminal resolution site (TRS) and Rep binding site. The ceDNA vector is preferably double-stranded, e.g., self-complementary, over at least a portion of the molecule, such as an expression cassette (e.g., the ceDNA is not a double-stranded circular molecule). The ceDNA vectors have covalently closed ends and are therefore resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III) at 37°C for, for example, 1 hour or more.

[0167] In one aspect of any of the aspects or embodiments herein, the ceDNA vector comprises, in a 5' to 3' direction, a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR. In one embodiment of any of the aspects or embodiments herein, the first ITR (5'ITR) and the second ITR (3'ITR) are asymmetric with respect to each other. That is, they have different three-dimensional spatial configurations. As an exemplary embodiment, the first ITR can be a wild-type ITR and the second ITR can be a mutated or modified ITR, or vice versa, where the first ITR can be a mutated or modified ITR and the second ITR can be a wild-type ITR. In one embodiment of any of the aspects or embodiments herein, the first ITR and the second ITR are both modified but have different sequences, different modifications, or are not identical modified ITRs and have different three-dimensional spatial configurations. In other words, ceDNA vectors using asymmetric ITRs have ITRs in which any changes in one ITR relative to the WT-ITR are not reflected in the other ITR, or alternatively, the asymmetric ITRs may have modified asymmetric ITR pairs, which may have different sequences and different three-dimensional shapes relative to each other.

[0168] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises, from 5' to 3', a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest (e.g., an expression cassette described herein), and a second AAV ITR, wherein the first ITR (5'ITR) and the second ITR (3'ITR) are symmetrical or substantially symmetrical with respect to each other; i.e., the ceDNA vector may comprise ITR sequences with a symmetrical three-dimensional spatial organization, such that their structures are the same shape in geometric space or have the same A, C-C', and B-B' loops in three-dimensional space. In such embodiments, the symmetrical or substantially symmetrical ITR pair may be modified ITRs (e.g., mod-ITRs) that are not wild-type ITRs. A mod-ITR pair may have one or more modifications from the wild-type ITR and have the same sequence that is the reverse complement (inverted) of each other. In one embodiment of any of the aspects or embodiments herein, the modified ITR pair is substantially symmetrical as defined herein, i.e., the modified ITR pair may have different sequences but may have corresponding or the same symmetrical three-dimensional shapes. In some embodiments of any of the aspects and embodiments herein, the symmetrical or substantially symmetrical ITRs may be wild-type (WT-ITR) as described herein. That is, both ITRs have wild-type sequences, but are not necessarily WT-ITRs of the same AAV serotype. In one embodiment of any of the aspects or embodiments herein, one WT-ITR may be derived from one AAV serotype, and the other WT-ITR may be derived from a different AAV serotype. In such embodiments, the WT-ITR pair is substantially symmetrical as defined herein, i.e., they may have one or more conservative nucleotide modifications while maintaining a symmetrical three-dimensional spatial configuration.

[0169] The wild-type or mutant or otherwise modified ITR sequences provided herein represent DNA sequences included in expression constructs (e.g., ceDNA-plasmids, ceDNA-bacmids, ceDNA-baculoviruses) for the production of ceDNA vectors. Thus, the ITR sequences actually contained in ceDNA vectors produced from ceDNA-plasmids or other expression constructs may or may not be identical to the ITR sequences provided herein as a result of naturally occurring variations (e.g., replication errors) that occur during the production process.

[0170] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector described herein, which comprises an expression cassette having a transgene that is a therapeutic nucleic acid sequence, can be operably linked to one or more regulatory sequences that enable or control expression of the transgene. In one embodiment of any of the aspects or embodiments herein, the polynucleotide comprises a first ITR sequence and a second ITR sequence, the nucleotide sequence of interest is flanked by the first and second ITR sequences, and the first and second ITR sequences are asymmetric with respect to each other or symmetric with respect to each other.

[0171] In one embodiment of any of the aspects or embodiments herein, the expression cassette is located between two ITRs and comprises, in that order, a promoter operably linked to a transgene, a post-transcriptional regulatory element, and one or more polyadenylation and termination signals. In one embodiment of any of the aspects or embodiments herein, the promoter is regulatable—inducible or repressible. The promoter can be any sequence that promotes transcription of the transgene. In one embodiment of any of the aspects or embodiments herein, the promoter is a CAG promoter or a variant thereof. The post-transcriptional regulatory element is a sequence that regulates expression of the transgene, and by way of non-limiting example, is any sequence that creates a tertiary structure that enhances expression of the transgene, which is a therapeutic nucleic acid sequence.

[0172] In one embodiment of any of the aspects or embodiments herein, the post-transcriptional regulatory element comprises a WPRE. In one embodiment of any of the aspects or embodiments herein, the polyadenylation and termination signal comprises a BGH polyA. Any cis-regulatory element known in the art, or combinations thereof, may additionally be used, such as the SV40 late polyA signal upstream enhancer sequence (USE) or other post-transcriptional processing elements (including, but not limited to, the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV)). In one embodiment of any of the aspects or embodiments herein, the length of the expression cassette in the 5' to 3' direction exceeds the maximum length known to be encapsidated in AAV virions. In one embodiment of any of the aspects or embodiments herein, the length is greater than 4.6 kb, or greater than 5 kb, or greater than 6 kb, or greater than 7 kb. Various expression cassettes are exemplified herein.

[0173] In one embodiment of any of the aspects or embodiments herein, the expression cassette may comprise more than 4000 nucleotides, 5000 nucleotides, 10,000 nucleotides, or 20,000 nucleotides, or 30,000 nucleotides, or 40,000 nucleotides, or 50,000 nucleotides, or any range from about 4000 to 10,000 nucleotides, or 10,000 to 50,000 nucleotides, or more than 50,000 nucleotides.

[0174] In one embodiment of any of the aspects or embodiments herein, the expression cassette may also include an internal ribosome entry site (IRES) and / or a 2A element. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments of any of the aspects and embodiments herein, the ITRs may act as promoters for the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may include additional components for regulating expression of the transgene, such as a regulatory switch for controlling and regulating expression of the transgene, and, if desired, a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0175] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is capsid-free and may be obtained from a plasmid encoding, in that order, a first ITR, an expressible transgene cassette, and a second ITR, wherein at least one of the first and / or second ITR sequences is mutated with respect to the corresponding wild-type AAV2 ITR sequence.

[0176] In one embodiment of any of the aspects or embodiments herein, the ceDNA vectors disclosed herein are used for therapeutic purposes (e.g., medical, diagnostic, or veterinary uses) or immunogenic polypeptides.

[0177] The expression cassette can include any transgene that is a therapeutic nucleic acid sequence. In certain embodiments, the ceDNA vector includes any gene of interest in a subject, including one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen-binding fragments, or any combination thereof.

[0178] In one embodiment of any of the aspects or embodiments herein, the sequences provided in the expression cassettes, expression constructs, or donor sequences of the ceDNA vectors described herein can be codon-optimized for the host cell. As used herein, the term "optimized codons" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, two or more, or a substantial number of codons of the native sequence (e.g., a prokaryotic sequence) with codons more frequently or most frequently used in the genes of a vertebrate of interest, such as a mouse or human, for enhanced expression in the cells of that vertebrate. Different species exhibit particular biases for certain codons for particular amino acids.

[0179] Typically, codon optimization does not change the amino acid sequence of the original translated protein. Optimized codons can be determined, for example, using Aptagen's Gene Forge® codon optimization and custom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite 300, Herndon, Va. 20171) or another public database.

[0180] Many organisms exhibit a bias for using certain codons to encode the insertion of specific amino acids in growing peptide chains. Codon preference, or codon bias, is a difference in codon usage among organisms, resulting from the degeneracy of the genetic code and is well documented among many organisms. Codon bias is often correlated with the efficiency of messenger RNA (mRNA) translation and is thought to depend, in particular, on the properties of the codon being translated and the availability of specific transfer RNA (tRNA) molecules. The prevalence of selected tRNAs within a cell generally reflects the codons most frequently used in peptide synthesis. Therefore, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0181] Given the large number of gene sequences available in a wide variety of animal, plant, and microbial species, it is possible to calculate the relative frequencies of codon usage (Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000)).

[0182] inverted terminal repeat (ITR) As described herein, a ceDNA vector is a non-encapsid-containing linear double-stranded DNA molecule formed from continuous strands of complementary DNA with covalently linked ends (linear, continuous, non-encapsid structure), and contains 5' inverted terminal repeat (ITR) and 3' ITR sequences that are different or asymmetric with respect to each other. At least one of the ITRs contains a functional terminal resolution site and a replication protein binding site (RPS) (sometimes called a replication protein binding site), e.g., a Rep binding site. Generally, a ceDNA vector contains at least one modified AAV inverted terminal repeat (ITR), i.e., a deletion, insertion, and / or substitution relative to the other ITR, and an expressible transgene.

[0183] In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is an AAV ITR, e.g., a wild-type AAV ITR. In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a modified ITR relative to the other ITRs, i.e., the ceDNA comprises ITRs that are asymmetric with respect to each other. In one embodiment of any of the aspects or embodiments herein, at least one of the ITRs is a non-functional ITR.

[0184] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises (1) an expression cassette comprising a cis-regulatory element, a promoter, and at least one transgene, (2) the promoter operably linked to the at least one transgene, and (3) two self-complementary sequences, e.g., ITRs, flanking the expression cassette, wherein the ceDNA vector is not associated with a capsid protein. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector comprises two self-complementary sequences found in the AAV genome, at least one of which comprises an operational Rep-binding element (RBE) and an AAV terminal resolution site (TRS) or a functional variant of the RBE, and one or more cis-regulatory elements operably linked to the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may comprise an additional component for regulating expression of the transgene, e.g., a regulatory switch for controlling and regulating expression of the transgene, and may comprise a regulatory switch that is a kill switch that enables controlled cell death of cells containing the ceDNA vector.

[0185] In one embodiment of any of the aspects or embodiments herein, the two self-complementary sequences can be ITR sequences from any known parvovirus, e.g., a dependovirus such as AAV (e.g., AAV1-AAV12). Any AAV serotype can be used, including, but not limited to, a modified AAV2 ITR sequence that retains a Rep binding site (RBS) and a terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. In some embodiments of any of the aspects and embodiments herein, the ITRs can be synthetic. In one embodiment of any of the aspects or embodiments herein, the synthetic ITRs are based on ITR sequences from two or more AAV serotypes. In another embodiment, the synthetic ITRs do not contain AAV base sequences. In yet another embodiment, the synthetic ITRs preserve the ITR structure described above but have little or no AAV-origin sequences. In some aspects, the synthetic ITRs may preferentially interact with wild-type Rep or Rep of a particular serotype, or in some cases, are not recognized by wild-type Rep but are recognized only by mutant Rep. In some embodiments of any of the aspects and embodiments herein, the ITRs are synthetic ITR sequences that retain a functional Rep binding site (RBS) and terminal resolution site (TRS), such as 5'-GCGCGCTCGCTCGCTC-3', in addition to a variable palindromic sequence that allows for hairpin secondary structure formation. In some examples, the modified ITR sequence retains the sequences of the RBS, TRS, and the structure and position of the Rep binding element that forms the terminal loop portion of one ITR hairpin secondary structure from the corresponding sequence of the wild-type AAV2 ITR. Exemplary ITR sequences for use in ceDNA vectors are disclosed in Tables 2-9, 10A and 10B, SEQ ID NOs: 2, 52, 101-449 and 545-547, and the partial ITR sequences shown in Figures 26A-26B of PCT International Application No. PCT / US18 / 49996, filed September 7, 2018.In some embodiments of any of the aspects and embodiments herein, the ceDNA vector may comprise an ITR with a modification in the ITR that corresponds to any of the modifications in the ITR sequence or ITR subsequence set forth in any one or more of Tables 2, 3, 4, 5, 6, 7, 8, 9, 10A and 10B of PCT International Application No. PCT / US18 / 49996, filed September 7, 2018.

[0186] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector can be produced from an expression construct that further comprises a specific combination of cis-regulatory elements. Cis-regulatory elements include, but are not limited to, promoters, riboswitches, insulators, mir regulatory elements, post-transcriptional regulatory elements, tissue- and cell-type-specific promoters, and enhancers. In some embodiments of any of the aspects and embodiments herein, the ITRs can act as promoters for the transgene. In some embodiments of any of the aspects and embodiments herein, the ceDNA vector comprises an additional component for regulating expression of the transgene, such as a regulatory switch described in PCT International Application No. PCT / US18 / 49996, filed September 7, 2018, to regulate expression of the transgene or a kill switch that can kill cells containing the ceDNA vector.

[0187] In one embodiment of any of the aspects or embodiments herein, the expression cassette may also include a post-transcriptional element to increase transgene expression. In one embodiment of any of the aspects or embodiments herein, the woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE) is used to increase transgene expression. Other post-transcriptional processing elements, such as post-transcriptional elements from the thymidine kinase gene of herpes simplex virus or hepatitis B virus (HBV), can be used. A secretory sequence may be linked to the transgene, for example, the VH-02 and VK-A26 sequences. The expression cassette may include a polyadenylation sequence known in the art, such as a natural sequence isolated from bovine BGHpA or viral SV40pA, or a synthetic sequence, or a variant thereof. Some expression cassettes may also include an SV40 late polyA signal upstream enhancer (USE) sequence. The USE may be used in combination with SV40pA or a heterologous polyA signal.

[0188] Figures 1A-1C of International Application No. PCT / US2018 / 050042, filed September 7, 2018, and incorporated herein by reference in its entirety, show schematic diagrams of non-limiting exemplary ceDNA vectors, or corresponding sequences of ceDNA plasmids. The ceDNA vectors are capsid-free and can be obtained from plasmids encoding, in that order, a first ITR, an expressible transgene cassette, and a second ITR, where at least one of the first and / or second ITR sequences is mutated relative to the corresponding wild-type AAV2 ITR sequence. The expressible transgene cassette preferably includes, in that order, one or more of an enhancer / promoter, an ORF reporter (transgene), a post-transcriptional regulatory element (e.g., WPRE), and a polyadenylation and termination signal (e.g., BGH polyA).

[0189] promoter Suitable promoters, including those mentioned above, can be derived from viruses and therefore can be referred to as viral promoters, or they can be derived from any organism, including prokaryotes or eukaryotes. Suitable promoters can be used to direct the expression of any RNA polymerase (e.g., pol Expression can be driven by SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVTE), Rous sarcoma virus (RSV) promoter, human U6 micronucleus promoter (U6, e.g., (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1;31(17)), human H1 promoter (H1), CAG promoter, human alpha 1-antitrypsin (HAAT) promoter (e.g., etc.). In one embodiment of any of the aspects or embodiments herein, these promoters are modified at their downstream intron-containing ends to contain one or more nuclease cleavage sites. In one embodiment of any of the aspects or embodiments herein, the DNA containing the nuclease cleavage sites is exogenous to the promoter DNA.

[0190] In one embodiment of any of the aspects or embodiments herein, the promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or alter its spatial and / or temporal expression. The promoter can also include distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the start site of transcription. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can regulate the expression of genetic components constitutively or differentially with respect to the cell, tissue, or organ in which expression occurs, with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter, or SV40 late promoter and CMV IE promoter, as well as the promoters listed below. Such promoters and / or enhancers can be used for expression of any gene of interest, e.g., a therapeutic protein. For example, a vector can include a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. In one embodiment of any of the aspects or embodiments herein, the promoter operably linked to the therapeutic protein coding sequence can be a promoter from Simian Virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, e.g., the long terminal repeat (LTR) promoter of bovine immunodeficiency virus (BIV), a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter, e.g., a CMV immediate early promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.In one embodiment of any of the aspects or embodiments herein, the promoter may also be a promoter from a human gene such as human ubiquitin C (hUbC), human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter may also be a natural or synthetic tissue-specific promoter, e.g., a liver-specific promoter, e.g., human alpha 1-antitrypsin (HAAT) or transthyretin (TTR). In one embodiment of any of the aspects or embodiments herein, delivery to the liver may be achieved using endogenous ApoE-specific targeting of a composition comprising a ceDNA vector to hepatocytes via the low-density lipoprotein (LDL) receptor present on the surface of hepatocytes.

[0191] In one embodiment of any of the aspects or embodiments herein, the promoter used is the native promoter of the gene encoding the therapeutic protein. The promoter and other regulatory sequences of each gene encoding the therapeutic protein are known and characterized. The promoter region used may further include one or more additional regulatory sequences (e.g., native), such as enhancers known in the art (e.g., Serpin enhancers).

[0192] Non-limiting examples of suitable promoters for use in accordance with the present invention include, for example, the CAG promoter, the HAAT promoter, the human EF1-α promoter, or fragments of the EF1-α promoter and the rat EF1-α promoter.

[0193] Polyadenylation sequence A sequence encoding a polyadenylation sequence can be included in a ceDNA vector to stabilize mRNA expressed from the ceDNA vector and to aid in nuclear transport and translation. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not contain a polyadenylation sequence. In other embodiments, the vector contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 45, at least 50, or more adenine dinucleotides. In some embodiments of any of the aspects and embodiments herein, the polyadenylation sequence contains about 43 nucleotides, about 40-50 nucleotides, about 40-55 nucleotides, about 45-50 nucleotides, about 35-50 nucleotides, or any range therebetween.

[0194] In one embodiment of any of the aspects or embodiments herein, the ceDNA is obtainable from a vector polynucleotide encoding a heterologous nucleic acid operably positioned between two different inverted terminal repeats (ITRs) (e.g., AAV ITRs), at least one of the ITRs comprising a terminal resolution site and a replication protein binding site (RPS), e.g., a Rep binding site (e.g., a wt AAV ITR), and one of the ITRs comprising a deletion, insertion, and / or substitution relative to the other ITR, e.g., a functional ITR.

[0195] In one embodiment of any of the aspects or embodiments herein, the host cell does not express viral capsid proteins, and the polynucleotide vector template lacks any viral capsid-encoding sequence. In one embodiment of any of the aspects or embodiments herein, the polynucleotide vector template lacks AAV capsid genes, but also lacks capsid genes of other viruses). In one embodiment of any of the aspects or embodiments herein, the nucleic acid molecule also lacks AAV Rep protein-encoding sequence. Thus, in some embodiments of any of the aspects and embodiments herein, the nucleic acid molecule of the invention lacks both functional AAV cap and AAV rep genes.

[0196] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector does not have modified ITRs.

[0197] In one embodiment of any of the aspects or embodiments herein, the ceDNA vector comprises a regulatory switch disclosed herein (or in PCT Application No. PCT / US18 / 49996, filed September 7, 2018).

[0198] IV. Production of ceDNA Vectors Methods for producing the ceDNA vectors described herein, which include asymmetric or symmetric ITR pairs as defined herein, are described in Section IV of PCT / US18 / 49996, filed September 7, 2018, which is incorporated herein by reference in its entirety. As described herein, ceDNA vectors can be obtained, for example, by a process comprising: a) incubating a population of host cells (e.g., insect cells) harboring a polynucleotide expression construct template (e.g., a ceDNA-plasmid, a ceDNA-bacmid, and / or a ceDNA-baculovirus), wherein the host cells lack viral capsid-coding sequences, under conditions effective and for a time sufficient to induce production of the ceDNA vector in the host cells in the presence of Rep proteins; and b) harvesting and isolating the ceDNA vector from the host cells. The presence of Rep proteins induces replication of the vector polynucleotide with the modified ITRs to produce the ceDNA vector in the host cells.

[0199] However, viral particles (e.g., AAV virions) are not expressed, and therefore there are no size limitations such as those naturally imposed on AAV or other virus-based vectors.

[0200] The presence of a ceDNA vector isolated from a host cell can be confirmed by digesting the DNA isolated from the host cell with a restriction enzyme that has a single recognition site on the ceDNA vector and analyzing the digested DNA material on a non-denaturing gel to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0201] In one embodiment of any of the aspects or embodiments herein, the invention provides for the use of host cell lines that have stably integrated a DNA vector polynucleotide expression template (ceDNA template) into their own genome in the production of non-viral DNA vectors, e.g., as described in Lee, L. et al. (2013) Plos One 8(8):e69879. Preferably, Rep is added to the host cells at an MOI of about 3. When the host cell line is a mammalian cell line, e.g., HEK293 cells, the cell line may have a stably integrated polynucleotide vector template, and a second vector, such as a herpesvirus, may be used to introduce Rep proteins into the cells, allowing for excision and amplification of the ceDNA in the presence of Rep and a helper virus.

[0202] In one embodiment of any of the aspects or embodiments herein, the host cells used to generate the ceDNA vectors described herein are insect cells, and baculovirus is used to deliver both the polynucleotide encoding the Rep protein and the non-viral DNA vector polynucleotide expression construct template for the ceDNA. In some embodiments of any of the aspects and embodiments herein, the host cells are engineered to express the Rep protein.

[0203] The ceDNA vector is then harvested and isolated from the host cells. The time for harvesting and collecting the ceDNA vector described herein from the cells can be selected and optimized to achieve high-yield production of the ceDNA vector. For example, the harvest time can be selected taking into account cell viability, cell morphology, cell growth, etc. In one embodiment of any of the aspects or embodiments herein, the cells are grown under sufficient conditions and harvested after sufficient time has passed since baculovirus infection to produce the ceDNA vector, but before most of the cells begin to die due to baculovirus toxicity. The DNA vector can be isolated using a plasmid purification kit, such as the Qiagen Endo-Free Plasmid Kit. Other methods developed for plasmid isolation can also be adapted for DNA vectors. Generally, any nucleic acid purification method can be employed.

[0204] The DNA vector can be purified by any means known to those skilled in the art for purifying DNA. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as a DNA molecule. In one embodiment of any of the aspects or embodiments herein, the ceDNA vector is purified as an exosome or microparticle. The presence of the ceDNA vector can be confirmed by digesting vector DNA isolated from cells with a restriction enzyme that has a single recognition site on the DNA vector, and analyzing both the digested and undigested DNA material using gel electrophoresis to confirm the presence of a characteristic linear and continuous DNA band compared to linear and discontinuous DNA.

[0205] V. Lipid Particle Preparation Lipid particles (e.g., lipid nanoparticles) can form spontaneously upon mixing of TNA (e.g., ceDNA) and lipids. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nm cutoff) using a thermobarrel extruder, such as the Lipex Extruder (Northern Lipids, Inc.). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.

[0206] Generally, lipid particles (e.g., lipid nanoparticles) can be formed by any method known in the art. For example, lipid particles (e.g., lipid nanoparticles) can be prepared by methods described in, for example, US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129, and US2010 / 0130588, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments of any of the aspects and embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared using a continuous mixing method, a direct dilution process, or an in-line dilution process. Processes and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution processes are described in US2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. A process and apparatus for preparing lipid nanoparticles using a serial dilution process is described in US2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0207] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) can be prepared by an impinging jet process. Generally, the particles are formed by mixing lipids dissolved in alcohol (e.g., ethanol) with ceDNA dissolved in a buffer solution, such as citrate buffer, sodium acetate buffer, sodium acetate and magnesium chloride buffer, malic acid buffer, malic acid and sodium chloride buffer, or sodium citrate and sodium chloride buffer. The lipid to ceDNA ratio can be about 45-55% lipid and about 65-45% ceDNA.

[0208] The lipid solution can contain the disclosed ionizable lipids, non-cationic lipids (e.g., phospholipids such as DSPC, DOPE, and DOPC), PEG-lipid-conjugated molecules (e.g., PEG-lipids), and sterols (e.g., cholesterol) in an alcohol, e.g., ethanol, at a total lipid concentration of 5-30 mg / mL, more likely 5-15 mg / mL, and most likely 9-12 mg / mL. In the lipid solution, the molar ratio of lipids can range from about 25-98%, preferably about 35-65%, for cationic lipids; about 0-15%, preferably about 0-12%, for non-ionic lipids; about 0-15%, preferably about 1-6%, for PEG-lipid-conjugated lipid molecules; and about 0-75%, preferably about 30-50%, for sterols.

[0209] The ceDNA solution can contain ceDNA in a buffer solution having a pH in the range of 3.5 to 5, at a concentration in the range of 0.3 to 1.0 mg / mL, preferably 0.3 to 0.9 mg / mL.

[0210] To form LNPs, in one exemplary but non-limiting embodiment, two liquids are heated to a temperature in the range of about 15-40°C, preferably about 30-40°C, and then mixed, for example, in an impinging jet mixer to instantly form LNPs. The mixing flow rate can be in the range of 10-600 mL / min. The tube ID range can be 0.25-1.0 mm, and the total flow rate can be 10-600 mL / min. The combination of flow rate and tube ID can have the effect of controlling the particle size of the LNPs to 30-200 nm. The solution can then be mixed with a buffer solution at a higher pH in a mixing ratio ranging from 1:1 to 1:3 vol:vol, preferably about 1:2 vol:vol. Optionally, this buffer solution can be at a temperature in the range of 15-40°C or 30-40°C. The mixed LNPs can then undergo an anion exchange filtration step. Prior to anion exchange, the mixed LNPs can be incubated for a period of time, for example, 30 minutes to 2 hours. The temperature during incubation can range from 15 to 40 °C or from 30 to 40 °C. After incubation, filter the solution through a filter, such as a 0.8 µm filter, which includes an anion exchange separation step. Tube IDs ranging from 1 mm ID to 5 mm ID and flow rates of 10 to 2000 mL / min can be used for this process.

[0211] After formation, the LNPs can be concentrated and ultrafiltered via an ultrafiltration process in which the alcohol is removed and the buffer is exchanged for a final buffer solution, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

[0212] The ultrafiltration process can use a tangential flow filtration (TFF) format with membranes having a nominal molecular weight cutoff range of 30–500 kD. Membrane formats can be hollow fiber or flat-sheet cassettes. In a TFF process with an appropriate molecular weight cutoff, the LNPs can be retained in the retentate, while the filtrate or permeate contains alcohol, citrate buffer, and final buffer waste. The TFF process is a multi-step process that results in an initial ceDNA concentration of 1–3 mg / mL. After concentration, the LNP solution is ultrafiltered with 10–20 volumes of final buffer to remove alcohol and perform buffer exchange. The material can then be further concentrated 1–3 times. The concentrated LNP solution can be sterile filtered.

[0213] VI. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions comprising the TNA lipid particles and a pharmaceutically acceptable carrier or excipient.

[0214] In one embodiment of any of the aspects or embodiments herein, TNA lipid particles (e.g., lipid nanoparticles) are provided with complete or partial encapsulation of a therapeutic nucleic acid. In one embodiment of any of the aspects or embodiments herein, the nucleic acid therapeutic is completely encapsulated in the lipid particle (e.g., lipid nanoparticle) to form a nucleic acid containing lipid particle. In one embodiment of any of the aspects or embodiments herein, the nucleic acid may be encapsulated within the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0215] In one embodiment of any of the aspects or embodiments herein, the lipid particles are about 20 nm to about 100 nm, 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, The average diameter of the nucleic acid-containing lipid particles (e.g., lipid nanoparticles) is about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. Nucleic acid-containing lipid particles (e.g., lipid nanoparticles) and methods for their preparation are disclosed, for example, in PCT / US18 / 50042, U.S. Patent Publication Nos. 2004 / 0142025 and 2007 / 0042031, the disclosures of which are incorporated herein by reference in their entireties for all purposes. In one embodiment of any of the aspects or embodiments herein, lipid particle (e.g., lipid nanoparticle) size can be determined by quasi-elastic light scattering, for example, using a Malvern Zetasizer Nano ZS (Malvern, UK) system.

[0216] Generally, the lipid particles (eg, lipid nanoparticles) of the present invention have an average diameter selected to provide the intended therapeutic effect.

[0217] Depending on the intended use of the lipid particle (e.g., lipid nanoparticle), the proportions of the components may vary, and the delivery efficiency of a particular formulation may be measured using, for example, an endosomal release parameter (ERP) assay.

[0218] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) can be conjugated with other moieties to prevent aggregation.Such lipid conjugates include, but are not limited to, PEG-lipid conjugates such as dialkyloxypropyl conjugates (e.g., PEG-DAA conjugates), diacylglycerol conjugates (e.g., PEG-DAG conjugates), cholesterol conjugates, phosphatidylethanolamine conjugates, and ceramide conjugates (see, e.g., U.S. Patent No. 5,885,613), cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates, e.g., U.S. Provisional Application No. 61 / 294,828 filed January 13, 2010, and U.S. Provisional Application No. 61 / 295,140 filed January 14, 2010), polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. Additional examples of POZ-lipid conjugates are described in PCT International Publication No. 2010 / 006282. PEG or POZ can be directly conjugated to lipids or linked to lipids via a linker moiety. For example, any linker moiety suitable for linking PEG or POZ to lipids can be used, including non-ester-containing linker moieties 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.

[0219] In one embodiment of any of the aspects or embodiments herein, the ceDNA may be complexed with the lipid portion of the particle or encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle). In one embodiment of any of the aspects or embodiments herein, the ceDNA may be fully encapsulated in the lipid portion of the lipid particle (e.g., lipid nanoparticle), thereby protecting it from degradation by, for example, nucleases in aqueous solution. In one embodiment of any of the aspects or embodiments herein, the ceDNA in the lipid particle (e.g., lipid nanoparticle) is not substantially degraded after exposure of the lipid particle (e.g., lipid nanoparticle) to nucleases for at least about 20, 30, 45, or 60 minutes at 37°C. In some embodiments of any of the aspects or embodiments herein, the ceDNA in the lipid particles (e.g., lipid nanoparticles) is not substantially degraded after incubation of the particles in serum at 37°C for at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0220] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) are substantially non-toxic to a subject, e.g., a mammal, such as a human.

[0221] In one embodiment of any of the aspects or embodiments herein, a pharmaceutical composition comprising a therapeutic nucleic acid of the present disclosure may be formulated into a lipid particle (e.g., lipid nanoparticle). In some embodiments of any of the aspects and embodiments herein, the lipid particle comprising the therapeutic nucleic acid may be formed from the disclosed ionizable lipids. In some other embodiments, the lipid particle comprising the therapeutic nucleic acid may be formed from a non-cationic lipid. In a preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles formed from the disclosed ionizable lipids containing a therapeutic nucleic acid selected from the group consisting of mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), minicircle DNA, minigene, viral DNA (e.g., lentivirus or AAV genome) or non-viral synthetic DNA vector, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmid, bacmid, doggybone™ DNA vector, minimal immunologically defined gene expression (MIDGE) vector, non-viral ministring DNA vector (linear covalently closed DNA vector), or dumbbell-shaped DNA minimal vector ("dumbbell DNA").

[0222] In another preferred embodiment, the lipid particles of the present invention are nucleic acid-containing lipid particles, which are formed from non-cationic lipids and, optionally, conjugated lipids that prevent aggregation of the particles.

[0223] In one embodiment of any of the aspects or embodiments herein, the lipid particle formulation is an aqueous solution. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation is a lyophilized powder.

[0224] According to some aspects, the present disclosure provides lipid particle formulations further comprising one or more pharmaceutical excipients. In one embodiment of any of the aspects or embodiments herein, the lipid particle (e.g., lipid nanoparticle) formulation further comprises sucrose, Tris, trehalose, and / or glycine.

[0225] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) disclosed herein can be incorporated into a pharmaceutical composition suitable for administration to a subject for in vivo delivery to the subject's cells, tissues, or organs. Typically, the pharmaceutical composition comprises the TNA lipid particles (e.g., lipid nanoparticles) disclosed herein and a pharmaceutically acceptable carrier. In one embodiment of any of the aspects or embodiments herein, the TNA lipid particles (e.g., lipid nanoparticles) of the present disclosure can be incorporated into a pharmaceutical composition suitable for the desired route of therapeutic administration (e.g., parenteral administration). Passive tissue transduction via high-pressure intravenous or intra-arterial infusion, as well as intracellular injections such as intranuclear microinjections or intracytoplasmic injections, are also contemplated. Pharmaceutical compositions for therapeutic purposes can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high ceDNA vector concentrations. Sterile injectable solutions can be prepared by incorporating the ceDNA vector compound in the required amount in an appropriate buffer with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

[0226] The lipid particles disclosed herein can be incorporated into pharmaceutical compositions suitable for local, systemic, intra-amniotic, intra-thecal, intracranial, intra-arterial, intravenous, intralymphatic, intraperitoneal, subcutaneous, tracheal, intra-tissue (e.g., intramuscular, intracardiac, intrahepatic, intrarenal, intracerebral), intra-thecal, intravesical, conjunctival (e.g., extraorbital, intraorbital, retroorbital, intraretinal, subretinal, choroidal, subchoroidal, intrainterstitial, intracameral, and intravitreal), intracochlear, and mucosal (e.g., oral, rectal, nasal) administration. Passive tissue transduction via high-pressure intravenous or intra-arterial infusion is also contemplated, as are intracellular injections such as intranuclear microinjection or intracytoplasmic injection.

[0227] Pharmaceutically active compositions containing TNA lipid particles (e.g., lipid nanoparticles) can be formulated to deliver a transgene in a nucleic acid to recipient cells, resulting in therapeutic expression of the transgene therein. The composition can also include a pharmaceutically acceptable carrier.

[0228] Pharmaceutical compositions for therapeutic purposes are typically sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for high ceDNA vector concentration.A sterile injectable solution can be prepared by incorporating the required amount of ceDNA vector compound in an appropriate buffer with one or a combination of the above-listed ingredients as needed, and then filtering sterilization.

[0229] In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) are solid core particles having at least one lipid bilayer. In one embodiment of any of the aspects or embodiments herein, the lipid particles (e.g., lipid nanoparticles) have a non-bilayer structure, i.e., a non-lamellar (i.e., non-bilayer) morphology. Without limitation, non-bilayer morphologies can include, for example, three-dimensional tubular, rod, cubic symmetry, etc. The non-lamellar morphology (i.e., non-bilayer structure) of lipid particles (e.g., lipid nanoparticles) can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryo-transmission electron microscopy ("Cryo-TEM"), differential scanning calorimetry ("DSC"), X-ray diffraction, etc. For example, the morphology (lamellar vs. non-lamellar) of lipid particles can be easily assessed and characterized using, for example, Cryo-TEM analysis as described in US2010 / 0130588 (the contents of which are incorporated herein by reference in their entirety).

[0230] In one embodiment of any of the aspects or embodiments herein, lipid particles (e.g., lipid nanoparticles) having a non-lamellar morphology are electron dense.

[0231] In one embodiment of any of the aspects or embodiments herein, the present disclosure provides lipid particles (e.g., lipid nanoparticles) that are either unilamellar or multilamellar in structure. In some aspects, the present disclosure provides lipid particle (e.g., lipid nanoparticle) formulations that include multivesicular particles and / or effervescent base particles. By controlling the composition and concentration of the lipid components, the rate at which lipid conjugates exchange out of the lipid particle (lipid nanoparticle), which in turn, the rate at which the lipid nanoparticle becomes fusogenic, can be controlled. In addition, other variables, including, for example, pH, temperature, or ionic strength, can be used to vary and / or control the rate at which lipid particles (e.g., lipid nanoparticles) become fusogenic. Other methods that can be used to control the rate at which lipid particles (e.g., lipid nanoparticles) become fusogenic will be apparent to those skilled in the art based on the present disclosure. It will also be apparent that lipid particle size can be controlled by controlling the composition and concentration of the lipid conjugate.

[0232] In one embodiment of any of the aspects or embodiments herein, the pKa of the formulated ionizable lipids can be correlated with the efficacy of the LNP for delivery of nucleic acids (Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533, Simple (See, e.g., J. Chem. Soc., 1999, 14:172-176 (2010), both of which are incorporated herein by reference in their entireties.) In one embodiment of any of the aspects or embodiments herein, the preferred range of pKa is about 5 to about 8. In one embodiment of any of the aspects or embodiments herein, the preferred range of pKa is about 6 to about 7. In one embodiment of any of the aspects or embodiments herein, the preferred pKa is about 6.5. In one embodiment of any of the aspects or embodiments herein, the pKa of an ionizable lipid can be determined in a lipid particle (e.g., a lipid nanoparticle) using an assay based on the fluorescence of 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS).

[0233] In one embodiment of any of the aspects or embodiments herein, encapsulation of ceDNA in lipid particles (e.g., lipid nanoparticles) can be determined by performing a membrane-impermeable fluorescent dye exclusion assay, such as the Oligreen® assay or the PicoGreen® assay, which uses a dye that exhibits enhanced fluorescence when associated with nucleic acids. Generally, encapsulation is determined by adding the dye to the lipid particle formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon the addition of a small amount of nonionic surfactant. Detergent-mediated disruption of the lipid bilayer releases the encapsulated ceDNA, allowing it to interact with the membrane-impermeable dye. Encapsulation of ceDNA can be calculated as E = (Io - I) / Io, where I and Io refer to the fluorescence intensity before and after the addition of surfactant.

[0234] Unit Dose In one embodiment of any of the aspects or embodiments herein, the pharmaceutical composition may be presented in a unit dosage form. The unit dosage form will typically be adapted for one or more routes of administration of the pharmaceutical composition. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by inhalation. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by an inhaler. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by a nebulizer. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for administration by an aerosolizer. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for oral, buccal, or sublingual administration. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for intravenous, intramuscular, or subcutaneous administration. In some embodiments of any of the aspects and embodiments herein, the unit dosage form is adapted for intrathecal or intracerebroventricular administration. In some embodiments of any of the aspects and embodiments herein, the pharmaceutical composition is formulated for topical administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect.

[0235] VII. Treatment Methods The ionizable lipid compositions and methods described herein (e.g., the TNA lipid particles (e.g., lipid nanoparticles) described herein) can be used to introduce nucleic acid sequences (e.g., therapeutic nucleic acid sequences) into host cells. In one embodiment of any of the aspects or embodiments herein, introduction of a nucleic acid sequence into a host cell using a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) can be monitored with appropriate biomarkers from the treated patient to assess gene expression.

[0236] The LNP compositions provided herein can be used to deliver transgenes (nucleic acid sequences) for a variety of purposes. In one embodiment of any of the aspects or embodiments herein, the ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used in a variety of ways, including, for example, ex situ, in vitro, and in vivo applications, methodologies, diagnostic procedures, and / or gene therapy regimens.

[0237] Provided herein is a method of treating a disease or disorder in a subject, comprising introducing a therapeutically effective amount of a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein), optionally with a pharmaceutically acceptable carrier, into target cells (e.g., liver cells, muscle cells, kidney cells, nerve cells, or other diseased cell type) of the subject in need of treatment. The implemented TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) comprises a nucleotide sequence of interest useful for treating the disease. In particular, the TNA may comprise a desired exogenous DNA sequence operably linked to a control element capable of directing transcription of a desired polypeptide, protein, or oligonucleotide encoded by the exogenous DNA sequence when introduced into a subject. The TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) can be administered via any suitable route described herein and known in the art. In one embodiment of any of the aspects or embodiments herein, the target cells are in a human subject.

[0238] Provided herein are methods for providing a diagnostically or therapeutically effective amount of TNA LNPs (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a subject in need thereof, the method comprising providing an amount of TNA LNPs (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a cell, tissue, or organ of a subject in need thereof for a time effective to allow expression of a transgene from the TNA LNPs, thereby providing a diagnostically or therapeutically effective amount of a protein, peptide, or nucleic acid expressed by the TNA LNPs (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to the subject. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0239] Provided herein are methods for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, disorder, dysfunction, injury, abnormal condition, or trauma in a subject. Generally, the methods include at least the step of administering a TNA LNP (e.g., a ceDNA vector lipid particle (e.g., lipid nanoparticle) described herein) to a subject in need thereof in an amount and for a time sufficient to diagnose, prevent, treat, or ameliorate one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the subject. In one embodiment of any of the aspects or embodiments herein, the subject is a human.

[0240] Provided herein are methods for using TNA LNPs as a tool for treating one or more symptoms of a disease or disease state. There are several genetic diseases for which the defective gene is known, and they are typically divided into two classes: enzyme deficiency states, which are usually inherited recessively, and imbalance states, which may involve regulatory or structural proteins, but are typically, but not always, inherited dominantly. In the case of deficiency diseases, TNA LNPs are LNPs (e.g., the ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) can be used to deliver transgenes to carry normal genes into diseased tissues for replacement therapy, and in some embodiments of any of the aspects and embodiments herein, antisense mutations can be used to create animal models of disease. In the case of imbalance disease states, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to create a disease state in a model system, which can then be used to attempt to counteract the disease state. Thus, the TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) and methods disclosed herein enable the treatment of genetic diseases. As used herein, a disease state is treated by partially or fully repairing the deficiency or imbalance that causes the disease or makes it more severe.

[0241] Generally, any transgene can be delivered using TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) as described above to treat, prevent, or ameliorate symptoms associated with any disorder related to gene expression. Exemplary disease states include, but are not limited to, cystic fibrosis (and other lung diseases), hemophilia A, hemophilia B, thalassemia, anemia and other blood disorders, AIDS, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, epilepsy and other neurological disorders, cancer, diabetes, muscular dystrophies (e.g., Duchenne, Becker), Hurler's disease, adenosine deaminase deficiency, metabolic disorders, retinal degenerative diseases (and other eye diseases), mitochondriopathy (e.g., Leber's hereditary optic neuropathy (LHON), Leigh's syndrome, and subacute sclerosing encephalitis), myopathy (e.g., facioscapulohumeral myopathy (FSHD) and cardiomyopathies), diseases of solid organs (e.g., brain, liver, kidney, heart), and the like. In some embodiments of any of the aspects and embodiments herein, the ceDNA vectors as disclosed herein may be advantageously used in the treatment of individuals with metabolic disorders (e.g., ornithine transcarbamylase deficiency).

[0242] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs described herein can be used to treat, ameliorate, and / or prevent diseases or disorders caused by mutations in genes or gene products. Exemplary diseases or disorders that can be treated with TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles) described herein) include, but are not limited to, metabolic diseases or disorders (e.g., Fabry disease, Gaucher disease, phenylketonuria (PKU), glycogen storage diseases); urea cycle diseases or disorders (e.g., ornithine transcarbamylase (OTC) deficiency); lysosomal storage diseases or disorders (e.g., metachromatic leukodystrophy (MLD), mucopolysaccharidosis type II (MPSII, Hunter syndrome)); liver diseases or disorders (e.g., progressive familial intrahepatic cholestasis (PFIC)); blood diseases or disorders (e.g., hemophilia A and B, thalassemia, and anemia); cancers and tumors, and genetic diseases or disorders (e.g., cystic fibrosis).

[0243] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles) can be used to deliver heterologous nucleotide sequences in situations where it is desirable to regulate the expression level of a transgene (e.g., a transgene encoding a hormone or growth factor).

[0244] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to correct abnormal levels and / or function of a gene product (e.g., an absence or defect in a protein) that results in a disease or disorder. The TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can produce functional proteins and / or restore protein levels to alleviate or reduce symptoms or confer benefits resulting from a particular disease or disorder caused by the absence or defect in the protein. For example, treatment of OTC deficiency can be achieved by producing functional OTC enzyme. Treatment of hemophilia A and B can be achieved by correcting levels of factor VIII, factor IX, and factor X. Treatment of PKU can be achieved by correcting levels of phenylalanine hydroxylase enzyme. Treatment of Fabry disease or Gaucher disease can be achieved by producing functional alpha-galactosidase or beta-glucocerebrosidase, respectively. Treatment of MFD or MPSII can be achieved by producing functional arylsulfatase A or iduronate-2-sulfatase, respectively. Treatment of cystic fibrosis can be achieved by producing functional cystic fibrosis transmembrane conductance regulator. Treatment of glycogen storage disease can be achieved by restoring functional G6Pase enzyme function. Treatment of PFIC can be achieved by producing functional ATP8B1, ABCB11, ABCB4, or TJP2 genes.

[0245] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver RNA-based therapeutics to cells in vitro or in vivo. Examples of RNA-based therapeutics include, but are not limited to, mRNA, antisense RNA and oligonucleotides, ribozymes, aptamers, interfering RNA (RNAi), Dicer substrate dsRNA, short hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), and microRNA (miRNA). For example, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver antisense nucleic acids to cells in vitro or in vivo. For example, if the transgene is an RNAi molecule, expression of the antisense nucleic acid or RNAi in the target cell reduces the expression of a specific protein by the cell. Thus, a transgene that is an RNAi molecule or an antisense nucleic acid can be administered to reduce the expression of a specific protein in a subject in need thereof. Antisense nucleic acids can also be administered to cells in vitro to regulate cellular physiology, for example, to optimize a cell or tissue culture system.

[0246] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver DNA-based therapeutics to cells in vitro or in vivo. Examples of DNA-based therapeutics include, but are not limited to, minicircle DNA, minigenes, viral DNA (e.g., lentivirus or AAV genomes) or non-viral synthetic DNA vectors, closed-end linear double-stranded DNA (ceDNA / CELiD), plasmids, bacmids, doggybone™ DNA vectors, minimal immunologically defined gene expression (MIDGE)-vectors, non-viral ministring DNA vectors (linear covalently closed DNA vectors), or dumbbell-shaped DNA minimal vectors ("dumbbell DNA"). For example, in one embodiment of any of the aspects or embodiments herein, ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be used to deliver minicircles to cells in vitro or in vivo. For example, if the transgene is minicircle DNA, expression of the minicircle DNA in target cells reduces the expression of a specific protein by the cells. Therefore, a transgene that is a minicircle DNA can be administered to reduce the expression of a particular protein in a subject in need thereof. Minicircle DNA can also be administered to cells in vitro to regulate cell physiology, for example, to optimize cell or tissue culture systems.

[0247] In one embodiment of any of the aspects or embodiments herein, exemplary transgenes encoded by the TNA vector comprising the expression cassette include a gene encoding a lysosomal enzyme (e.g., hexosaminidase A associated with Tay-Sachs disease, or a gene encoding ... II-related iduronate sulfatase), erythropoietin, angiostatin, endostatin, superoxide dismutase, globin, leptin, catalase, tyrosine hydroxylase, as well as cytokines (e.g., interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), nerve growth factor (NGF), neurotrophic factor-3 and 4, brain-derived neurotrophic factor (BDNF), glial-derived growth factor (GDNF), transforming growth factor-a and -b, etc.), receptors (e.g., tumor necrosis factor receptor). In some exemplary embodiments, the transgene encodes a monoclonal antibody specific for one or more desired targets. In some exemplary embodiments, two or more transgenes are encoded by a ceDNA vector. In some exemplary embodiments, the transgene encodes a fusion protein comprising two different polypeptides of interest. In some embodiments of any of the aspects and embodiments herein, the transgene encodes an antibody, including a full-length antibody or an antibody fragment, as defined herein. In some embodiments of any of the aspects and embodiments herein, the antibody is an antigen-binding domain or immunoglobulin variable domain sequence, as defined herein. Other exemplary transgene sequences encode suicide gene products (thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, and tumor suppressor gene products.

[0248] Administration In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles described herein) can be administered to an organism for transduction of cells in vivo. In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA vector lipid particles) can be administered to an organism for transduction of cells ex vivo.

[0249] Generally, administration is by any of the routes commonly used to ultimately bring molecules into contact with blood or tissue cells. Suitable methods for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, certain routes may often provide a more immediate and effective response than others. Exemplary administration modes of TNA LNPs (e.g., ceDNA vector lipid particles) include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intradermal, intrauterine (or intraocular), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to the skeleton, diaphragm, and / or myocardium), intrapleural, intracerebral, and intraarterial), topical (e.g., to the skin and mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, myocardium, diaphragm, muscle, or brain).

[0250] Administration of TNA LNPs (e.g., ceDNA LNPs) such as ceDNA vectors can be to any site of a subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye. In one embodiment of any of the aspects or embodiments herein, administration of the ceDNA LNP can also be to a tumor (e.g., within or near a tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition to be treated, ameliorated, and / or prevented, as well as the properties of the particular ceDNA LNP being used. Additionally, ceDNA allows for the administration of two or more transgenes in a single vector or multiple ceDNA vectors (e.g., a ceDNA cocktail).

[0251] In one embodiment of any of the aspects or embodiments herein, administration of ceDNA LNPs to skeletal muscles includes, but is not limited to, administration to skeletal muscles of the limbs (e.g., upper arms, lower arms, upper limbs, and / or lower limbs), lower back, neck, head (e.g., tongue), pharynx, abdomen, pelvis / perineum, and / or fingers. ceDNA vectors (e.g., ceDNA vector lipid particles (e.g., lipid nanoparticles)) can be delivered to skeletal muscles by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (optionally, isolated limb perfusion of the legs and / or arms, see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, ceDNA LNPs are administered to a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., by intravenous or intra-arterial administration). In one embodiment of any of the aspects or embodiments herein, the ceDNA LNPs can be administered without the use of "hydrodynamic" techniques.

[0252] Administration of TNA LNPs (e.g., ceDNA LNPs) to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. TNA LNPs (e.g., ceDNA LNPs) can be delivered to the myocardium by intravenous administration, intra-arterial administration such as intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion. Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. Administration to smooth muscle can be by any suitable method, including intravenous administration, intra-arterial administration, and / or intraperitoneal administration. In one embodiment of any of the aspects or embodiments herein, administration can be to endothelial cells present in, near, and / or on smooth muscle.

[0253] In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) are administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (e.g., to treat, ameliorate, and / or prevent muscular dystrophy or heart disease (e.g., PAD or congestive heart failure)).

[0254] TNA LNPs (e.g., ceDNA LNPs) can be administered to the CNS (e.g., the brain or eye). TNA LNPs (e.g., ceDNA LNPs) may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (cerebrum including striatum, occipital lobe, temporal lobe, parietal lobe, and frontal lobe, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. LNPs (e.g., ceDNA LNPs) can also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve. TNA LNPs (e.g., ceDNA LNPs) can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture). TNA LNPs (e.g., ceDNA vector lipid particles) can also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0255] In one embodiment of any of the aspects or embodiments herein, the TNA LNPs (e.g., ceDNA LNPs) can be administered to the desired region of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intracerebroventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, and intramuscular delivery with retrograde delivery to motor neurons.

[0256] According to some embodiments of any of the aspects or embodiments herein, a TNA LNPs (e.g., ceDNA LNPs) are administered in a liquid formulation by direct injection (e.g., stereotactic injection) into the desired region or compartment in the CNS. According to other embodiments, TNA LNPs (e.g., ceDNA LNPs) can be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye can also be by topical application of liquid drops. As a further alternative, ceDNA vectors can be administered as solid sustained-release formulations (see, e.g., U.S. Patent No. 7,201,898, incorporated herein by reference in its entirety). In one embodiment of any of the aspects or embodiments herein, TNA LNPs (e.g., ceDNA LNPs) can be used for retrograde transport to treat, ameliorate, and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), etc.). For example, TNA LNPs (e.g., ceDNA LNPs) can be delivered to muscle tissue and translocate from there into neurons.

[0257] In one embodiment of any of the aspects or embodiments herein, repeated administration of a therapeutic product can be performed until an appropriate level of expression is achieved. Thus, in one embodiment of any of the aspects or embodiments herein, a therapeutic nucleic acid can be administered and re-administered multiple times. For example, a therapeutic nucleic acid can be administered on day 0. Following the initial treatment on day 0, the therapeutic nucleic acid can be administered at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, or about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, or about 18 years after the initial treatment with the therapeutic nucleic acid. A second administration (re-administration) can be administered after about 18 years, about 19 years, about 20 years, about 21 years, about 22 years, about 23 years, about 24 years, about 25 years, about 26 years, about 27 years, about 28 years, about 29 years, about 30 years, about 31 years, about 32 years, about 33 years, about 34 years, about 35 years, about 36 years, about 37 years, about 38 years, about 39 years, about 40 years, about 41 years, about 42 years, about 43 years, about 44 years, about 45 years, about 46 years, about 47 years, about 48 years, about 49 years, or about 50 years.

[0258] In one embodiment of any of the aspects or embodiments herein, one or more additional compounds may also be included. These compounds may be administered separately, or the additional compounds may be included in the lipid particles (e.g., lipid nanoparticles) of the present invention. In other words, the lipid particles (e.g., lipid nanoparticles) may contain other compounds in addition to TNA, or at least a second TNA different from the first one. Without limitation, other additional compounds may be selected from the group consisting of small or large organic or inorganic molecules, monosaccharides, disaccharides, trisaccharides, oligosaccharides, polysaccharides, peptides, proteins, peptide analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, extracts made from biological materials, or any combination thereof.

[0259] In one embodiment of any of the aspects or embodiments herein, the one or more additional compounds may be therapeutic agents. The therapeutic agent may be selected from any class suitable for therapeutic purposes. Thus, the therapeutic agent may be selected from any class suitable for therapeutic purposes. The therapeutic agent may be selected according to the desired therapeutic purpose and biological effect. For example, in one embodiment of any of the aspects or embodiments herein, if the TNA in the LNP is useful for treating cancer, the additional compound may be an anti-cancer agent (e.g., a chemotherapeutic agent, a targeted cancer therapy (including, but not limited to, a small molecule, an antibody, or an antibody-drug conjugate)). In one embodiment of any of the aspects or embodiments herein, if the LNP containing the TNA is useful for treating an infectious disease, the additional compound may be an antimicrobial agent (e.g., an antibiotic or an antiviral compound). In one embodiment of any of the aspects or embodiments herein, if the LNP containing the TNA is useful for treating an immune disease or disorder, the additional compound may be an anti-inflammatory agent (e.g., an anti-inflammatory agent). The additional compound can be a compound that modulates the immune response (e.g., an immunosuppressant, an immunostimulatory compound, or a compound that modulates one or more specific immune pathways). In one embodiment of any of the aspects or embodiments herein, different cocktails of different lipid particles containing different compounds, such as TNAs encoding different proteins or different compounds (e.g., therapeutic agents), can be used in the compositions and methods of the invention. In one embodiment of any of the aspects or embodiments herein, the additional compound is an immunomodulatory agent. For example, the additional compound is an immunosuppressant. In some embodiments of any of the aspects and embodiments herein, the additional compound is immunostimulatory. [Example]

[0260] The following examples are provided by way of illustration and not by way of limitation. It will be understood by those skilled in the art that ionizable lipids can be designed and synthesized using the general synthetic methods described below.

[0261] General Synthesis Ionizable lipids of Formula I were designed and synthesized using a similar synthetic methodology shown in Scheme 1 below. [ka]

[0262] Example 1: Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (Lipid 1) Synthesis of the cleavable, ionizable head group ((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-hydroxyphenyl)acetate) (7) Step-1 [ka] Synthesis of disulfanediylbis(ethane-2,1-diyl) dimethanesulfonate (2). Commercially available 2,2'-disulfanediylbis(ethan-1-ol) (1) (15 g, 97.2 mmol) was dissolved in acetonitrile (143 mL), followed by the addition of triethylamine (NEt3) (33.3 g, 328 mmol). Methanesulfonyl chloride (MSCl) (34.5 g, 300 mmol) was added dropwise to the reaction mixture at 0 °C. The resulting reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched by the addition of ethanol (EtOH) (39 mL), and the insoluble material was removed by filtration. The filtrate was partitioned between dichloromethane (DCM) (150 mL) and 10% sodium bicarbonate / water (150 mL). The organic layer was washed four times with 100 ml of water, dried over magnesium sulfate (MgSO4), and evaporated to give 2 as a brown oil (25 g, 81%) that solidified on standing. 1H-NMR (300 MHz, d-chloroform): δ 4.43–4.48 (t, 4H), 3.00–3.10 (m, 10H).

[0263] Step-2 [ka] Synthesis of 2,2'-((disulfanediylbis(ethane-2,1-diyl))bis(piperidin-1,4-diyl))bis(ethan-1-ol) (4). To a solution of 2 (12 g, 38.7 mmol) in acetonitrile (310 ml), potassium carbonate (KCO) (13.4 g, 96.6 mmol) was added, followed by 2-(piperidin-4-yl)ethan-1-ol (3) (20 g, 155 mmol). The resulting mixture was stirred at room temperature overnight, and then filtered to remove insoluble material. The filtrate was evaporated to dryness to give the crude product, which was dissolved in DCM (100 ml), washed twice with water (50 ml), dried over MgSO, and evaporated to give 4 as a yellow oil (11.8 g, 79%). 1 H-NMR (300MHz, d-chloroform): δ3.63-3.68(t, 4H), 2.78-2.90(m, 8H), 2.62-2.65(t, 4H), 1.94-2.02 (t, 4H), 1.70(s, 2H), 1.65-1.70(d, 4H), 1.27-1.48(t, 4H), 1.40-1.50(m, 2H), 1.23-1.27(m, 4H).

[0264] Step-3 [ka] Synthesis of 2-(4-((tert-butyldimethylsilyl)oxy)phenyl)acetic acid (5). To a stirred solution of 4-hydroxyphenylacetic acid (5a) (10 g, 65 mmol) in dimethylformamide (DMF) (40 mL) at 0 °C, NEt (10 g, 100 mmol) was added, followed by tert-butyldimethylsilyl chloride (TBSCl) (15 g, 100 mmol). The resulting reaction mixture was stirred overnight at room temperature and then treated with water (200 mL) and DCM (150 mL). The organic phase was separated. The aqueous phase was extracted with DCM (100 mL). The combined organic phase was washed with a saturated solution of sodium bicarbonate, brine, and dried over sodium sulfate (NaSO). The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0–10% methanol (MeOH) in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 5 (4.8 g, 27%) and the di-tert-butyldimethylsilyl ether (di-TBS) by-product (10.5 g, 42%). 1 H-NMR (300 MHz, d-chloroform): δ 7.12 (d, 2H), 6.78 (d, 2H), 3.56 (s, 2H), 0.97 (s, 9H), 0.18 (s, 6H). [ka]

[0265] Synthesis of ((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-((tert-butyldimethylsilyl)oxy)phenyl)acetate) (6). To a stirred solution of disulfide 4 (1.92 g, 5 mmol) from step 2 and phenylacetic acid 5 (3.4 g, 12.8 mmol) in DCM (100 ml) was added 4-dimethylaminopyridine (DMAP) (1.5 g, 12.5 mmol), followed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (2.4 g, 12.5 mmol). The resulting mixture was stirred overnight at room temperature and then washed with saturated sodium bicarbonate solution (200 ml), brine (150 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give 6 (4.1 g, 92%). 1 H-NMR (300MHz, d-chloroform): δ7.12 (d, 4H), 6.75 (d, 4H), 4.1 (t, 4H), 3.5 (s, 4H), 2.82 (m, 8) H), 2.62(m, 4H), 1.93(t, 4H), 1.61-1.45(m, 8H), 1.26(m, 6H), 0.97(s, 18H), 0.17(s, 4H).

[0266] Step-4 [ka] Synthesis of ((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl)bis(2-(4-hydroxyphenyl)acetate) (7). To a stirred solution of disulfide 6 (3.1 g, 3.6 mmol) in tetrahydrofuran (THF) (40 mL) was added hydrogen fluoride pyridine (1 mL, 3.8 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 2 h and then at room temperature for an additional 2 h. The reaction mixture was treated with a saturated solution of sodium bicarbonate (200 mL) and extracted with ethyl acetate (2 × 150 mL). The combined organic phases were washed with brine (100 mL), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatography using 0–10% MeOH in DCM as the eluent to afford the desired product 7 (1.92 g, 82%). 1 H-NMR (300MHz, d-chloroform): δ7.13 (d, 4H), 6.70 (d, 4H), 4.1 (t, 4H), 3.5 (s, 4H), 2.89 (m, 8H), 2.70 (m, 4H), 1.95 (t, 4H), 1.48 (m, 8H), 1.17 (m, 6H).

[0267] Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (10) [ka] Synthesis of 9-(heptadecan-9-yloxy)-9-oxononanoic acid (10). To a stirred solution of nonanedioic acid (8) (7.34 g, 39 mmol) and heptadecan-9-ol (8b) (5 g, 19 mmol) in dichloromethane (1000 mL) was added DMAP (2.37 g, 19 mmol), followed by EDCI (3 g, 19 mmol). The resulting mixture was stirred overnight at room temperature and then washed with 250 mL of 1 N HCl and 250 mL of water. The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0–10% MeOH in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 10 (6.2 g, 75%) as a white solid.1 H-NMR (300 MHz, d-chloroform): δ 4.80-4.90 (m, 1H), 2.25-2.34 (m, 4H), 1.55-1.70 (m, 4H), 1.40-1.50 (m, 4H), 1.20-1.40 (m, 30H), 0.84-0.90 (t, 3H).

[0268] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate [ka] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (11). To a stirred solution of disulfide 7 (580 mg, 0.9 mmol) produced in step 4 and acid 10 (422 mg, 0.99 mmol) in DMF (20 ml) was added DMAP (165 mg, 1.35 mmol) followed by EDCI (258 mg, 1.35 mmol). The resulting mixture was stirred at room temperature overnight, and then saturated sodium bicarbonate solution (50 ml) was added. The reaction mixture was extracted with dichloromethane (2 x 50 ml). The combined organic phase was washed with brine (30 ml), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as eluent to afford the desired product 11 (427 mg, 45%). 1H-NMR (300MHz, d-chloroform): δ7.27(d, 2H), 7.11(d, 2H), 7.03(d, 2H), 6.69(d, 2H), 4.85(m, 1H), 4.1(m, 4H), 3.56(s, 2H), 3.48(s, 2H), 2.92(d, 2H), 2.85-2.69(m, 12H), 2.71(t, 2H), 2.28(t, 2H), 1.95(t, 2H), 1.52-1.01(m, 53H), 0.85(m, 6H).

[0269] Synthesis of lipid 1 [ka] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (lipid 1). To a stirred solution of disulfide 11 (151 mg, 0.14 mmol) and oleic acid 12 (61 mg, 0.22 mmol) in dichloromethane (10 ml), DMAP (28 mg, 0.22 mmol) was added, followed by EDCI (42 mg, 0.22 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 1 (126 mg, 68%). 1 H-NMR (300MHz, d-chloroform): δ7.25(d, 4H), 7.01(d, 4H), 5.34(m, 2H), 4.86(m, 1H), 4.11(t, 4H), 3.58(s, 4H), 2.91-2.70( m, 8H), 2.62(m, 4H), 2.53(t, 4H), 2.28(t, 2H), 2.05-1.87(m, 8H), 1.78-1.46(m, 22H), 1.48-1.23(m, 54H), 0.86(t, 9H). MS[M+H]+ 1318.

[0270] Example 2: Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((9-(nonyloxy)-9-oxononanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (Lipid 3) Synthesis of 9-(nonyloxy)-9-oxononanoic acid (9) [ka] Synthesis of 9-(nonyloxy)-9-oxononanoic acid (9). To a stirred solution of nonanedioic acid (8) (13.2 g, 0.1 mol) and nonan-1-ol (8a) (7.2 g, 0.05 mol) in DCM (1000 mL) was added DMAP (6.1 g, 0.05 mol), followed by EDCI (7.7 g, 0.05 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1N hydrochloric acid (HCl) solution (500 mL) and water (500 mL). The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0–10% MeOH in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 9 (12.6 g, 81%) as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.03-4.07 (t, 2H), 2.28-2.34 (m, 4H), 1.58-1.63 (m, 6H), 1.26-1.32 (m, 18H), 0.85-0.87 (t, 3H).

[0271] Lipid 3 synthesis [ka] Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((9-(nonyloxy)-9-oxononanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (Lipid 3). To a stirred solution of disulfide 11 (stepwise synthesis described in Example 1) (150 mg, 0.14 mmol) and acid 9 (62 mg, 0.22 mmol) in dichloromethane (10 ml) was added DMAP (28 mg, 0.22 mmol) followed by EDCI (42 mg, 0.22 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 3 (114 mg, 60%). 1 H-NMR (300MHz, d-chloroform): δ7.28(d, 4H), 7.02(d, 4H), 4.86(m, 1H), 4.11(t, 4H), 4.04(t, 2H), 3.58(s, 4H), 2.93-2. 77(m, 8H), 2.63(m, 4H), 2.53(t, 4H), 2.28(m, 4H), 1.95(t, 4H), 1.85-1.47(m, 24H), 1.45-1.16(m, 54H), 0.86(t, 9H). MS[M+H] + 1350.

[0272] Example 3: Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((5-(nonyloxy)-5-oxopentanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (Lipid 2) [ka] To a stirred solution of disulfide 11 (stepwise synthesis described in Example 1) (150 mg, 0.14 mmol) and acid 9a (see synthesis described in Example 1 for acid 9, substituting commercially available glutaric acid as the starting material for nonan-1-ol (8a) to produce 9a) (57 mg, 0.22 mmol) in DCM (10 ml), DMAP (28 mg, 0.22 mmol) was added, followed by EDCI (42 mg, 0.22 mmol). The resulting mixture was stirred overnight at room temperature and then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Evaporation of the fractions containing the desired compound afforded lipid 2 (151 mg, 81%). 1 H-NMR (300MHz, d-chloroform): δ7.26(d, 4H), 7.01(d, 4H), 4.86(m, 1H), 4.10-4.02(t, 6H), 3.57(s, 4H), 3.01(d, 4H) , 2.83-2.72(m, 4H), 2.34-2.21(m, 14H), 2.15-1.91(m, 6H), 1.74-1.41(m, 12H), 1.39-1.16(m, 52H), 0.86(t, 9H). MS[M+H]+1293.

[0273] Example 4: Synthesis of 1-(heptadecan-9-yl) 9-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-((5-(nonyloxy)-5-oxopentanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)nonanedioate (Lipid 4) [ka] To a stirred solution of disulfide 7 (stepwise synthesis described in Example 1) (150 mg, 0.23 mmol) and compound 9 (synthesis described in Example 2) (146 mg, 0.46 mmol) in a mixture of dichloromethane (5 mL) and DMF (3 mL), DMAP (70 mg, 0.57 mmol) was added followed by EDCI (109 mg, 0.57 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 15 minutes and then at room temperature overnight. DCM (20 mL) was added, and the reaction mixture was washed with saturated sodium bicarbonate solution (20 mL), brine (20 mL), and dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Evaporation of the fractions containing the desired compound afforded lipid 4 (180 mg, 63%). 1 H-NMR (300MHz, d-chloroform): δ7.28(d, 4H), 7.02(d, 4H), 4.11(t, 4H), 4.04(t, 4H), 3.58(s, 4H), 2.93-2.67(m, 8H) , 2.63-2.55(m, 4H), 2.53(t, 4H), 2.29(t, 4H), 1.94(t, 4H), 1.85-1.47(m, 20H), 1.45-1.16(m, 42), 0.87(t, 6H). MS[M+H] + 1237.

[0274] Example 5: Synthesis of O'1,O1-((((((disulfanediylbis(ethane-2,1-diyl))bis(piperidine-1,4-diyl))bis(ethane-2,1-diyl))bis(oxy))bis(2-oxoethane-2,1-diyl))bis(4,1-phenylene))9,9'-di(heptadecan-9-yl)di(nonanedioate) (Lipid 5) [ka] To a stirred solution of disulfide 7 (stepwise synthesis described in Example 1) (580 mg, 0.9 mmol) and acid 10 (synthesis described in Example 1) (422 mg, 0.99 mmol) in DMF (20 ml) was added DMAP (164 mg, 1.35 mmol) followed by EDCI (257 mg, 1.35 mmol) at 0 °C. The resulting mixture was stirred at 0 °C for 15 min and then at room temperature overnight. DCM (60 ml) was added, and the reaction mixture was washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Evaporation of the fractions containing the desired compound gave lipid 5 (280 mg, 38%). 1 H-NMR (300MHz, d-chloroform): δ7.26(d, 4H), 7.02(d, 4H), 4.85(m, 2H), 4.11(t, 4H), 3.58(s, 4H), 2.86-2.77(m, 8 H), 2.63(m, 4H), 2.53(t, 4H), 2.27(t, 4H), 1.92(t, 4H), 1.75-1.47(m, 26H), 1.45-1.16(m, 64H), 0.86(t, 12H). MS[M+H] + 1462.

[0275] Example 6: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(undecan-3-yl)nonanedioate (Lipid 6) Synthesis of 9-oxo-9-(undecan-3-yloxy)nonanoic acid (9b) [ka] Synthesis of 9-oxo-9-(undecan-3-yloxy)nonanoic acid (9b). To a stirred solution of nonanedioic acid (8) (10.9 g, 0.058 mol) and undecan-3-ol (8b) (5 g, 0.029 mol) in DCM (500 mL) was added DMAP (3.5 g, 0.03 mol), followed by EDCI (4.5 g, 0.03 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1N HCl solution (500 mL) and water (500 mL). The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 9b (6.5 g, 66%) as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.79-4.83 (t, 1H), 2.28-2.34 (m, 4H), 1.25-1.33 (m, 8H), 1.26-1.32 (m, 18H), 0.85-0.87 (t, 6H).

[0276] Synthesis of 4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyloleate (13) [ka] Synthesis of 4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl oleate (13). To a stirred solution of disulfide 7 (stepwise synthesis described in Example 1) (2.0 g, 3 mmol) and oleic acid (or acid 12 described in Example 1) (0.79 g, 2.8 mmol) in DCM (200 ml) was added DMAP (340 mg, 2.8 mmol), followed by EDCI (440 mg, 2.8 mmol). The resulting mixture was stirred at room temperature overnight, and then saturated sodium bicarbonate solution (20 ml) was added. The reaction mixture was extracted with dichloromethane (2 x 50 ml). The combined organic phase was washed with brine (30 ml), dried over NaSO, and concentrated. The residue was purified by column chromatography using 0-5% methanol in dichloromethane as the eluent to give 13 (1.6 g, 57%) as a white solid. The crude product was used directly in the next step without further characterization.

[0277] Synthesis of lipid 6 [ka] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(undecan-3-yl)nonanedioate (lipid 6). To a stirred solution of disulfide 13 (250 mg, 0.27 mmol) and acid 9b (113 mg, 0.33 mmol) in DCM (20 ml) was added DMAP (40 mg, 0.33 mmol), followed by EDCI (51 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 6 (120 mg, 36%). 1 H-NMR (300MHz, d-chloroform): δ7.31 (d, 4H), 7.05 (d, 4H), 5.36-5.40 (m, 2H), 4.86 (m, 1H), 4.11 (t, 4H), 3.62 (t, 4H), 2.77-2.90 (m, 8H), 2.55-2.71(m, 8H), 2.30-2.34(m, 2H), 1.96-2.05(m, 8H), 1.77(m, 4H), 1.58-1.67(m, 18H), 1.30-1.58(m, 40H), 0.89(t, 9H).

[0278] Example 7: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(tridecan-5-yl)nonanedioate (Lipid 7) Synthesis of tridecanol-5-ol (8c) [ka] Synthesis of tridecan-5-ol (8c). To a solution of aldehyde 8c-1 (7.1 g, 0.05 mol) in 100 ml of anhydrous THF was added dropwise a solution of 2 M butyllithium (BuLi) (27 ml) in THF at −78°C. The resulting mixture was stirred at −78°C for 2 h and then at room temperature for 2 h. The reaction was quenched by adding water and partitioned between 1 N HCl and ether. The organic layer was collected, dried over MgSO4, and evaporated to give crude 8c (10 g, 100%) as a yellow oil, which was used directly in the next step without further purification.

[0279] Synthesis of 9-oxo-9-(tridecan-5-yloxy)nonanoic acid (9c) [ka] Synthesis of 9-oxo-9-(tridecan-5-yloxy)nonanoic acid (9c). To a stirred solution of nonanedioic acid (8) (9.4 g, 0.05 mol) and 8c (5 g, 0.025 mol) in DCM (500 mL) was added DMAP (3.05 g, 0.025 mol), followed by EDCI (3.88 g, 0.025 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1N HCl solution (500 mL) and water (500 mL). The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 9c (2.5 g, 27%) as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.84-4.87 (t, 1H), 2.28-2.34 (m, 4H), 1.58-1.63 (m, 7H), 1.26-1.32 (m, 23H), 0.85-0.87 (t, 6H).

[0280] Synthesis of lipid 7 [ka] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(tridecan-5-yl)nonanedioate (Lipid 7). To a stirred solution of disulfide 13 (synthesis described in Example 6) (250 mg, 0.27 mmol) and acid 9c (116 mg, 0.33 mmol) in DCM (20 ml) was added DMAP (40 mg, 0.33 mmol), followed by EDCI (51 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 7 (160 mg, 40%). 1 H-NMR (300MHz, d-chloroform): δ7.29(d, 4H), 7.04(d, 4H), 5.29-5.34(m, 2H), 4.86(m, 1H), 4.11(t, 4H), 3.58(t, 4H), 2.77-2.90(m, 8H), 2.51-2.79(m, 8H), 2.28(m, 2H), 1.94-2.05(m, 8H), 1.70-1.80(m, 4H), 1.49-1.67(m, 18H), 1.10-1.40(m, 46H), 0.88(t, 9H).

[0281] Example 8: Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(pentadecan-7-yl)nonanedioate (Lipid 8) Synthesis of pentadecan-7-ol (8d) [ka] Synthesis of pentadecan-7-ol (8d). To a solution of aldehyde 8d-1 (7.1 g, 0.05 mol) in 100 ml of anhydrous THF was added a solution of 2 M hexylmagnesium bromide in THF (27 ml) at −78° C. The resulting mixture was stirred at −78° C. for 2 hours and then at room temperature overnight. The reaction was quenched by adding water and partitioned between 1 N HCl and ether. The organic layer was collected, dried over MgSO4, and evaporated to give crude 8d (11 g, 100%) as a white solid, which was used directly in the next step without further purification.

[0282] Synthesis of 9-oxo-9-(pentadecan-7-yloxy)nonanoic acid (9d) [ka] Synthesis of 9-oxo-9-(pentadecan-7-yloxy)nonanoic acid (9d). To a stirred solution of nonanedioic acid (8) (9.4 g, 0.05 mol) and pentadecan-7-ol (8d) (5.7 g, 0.025 mol) in DCM (1000 mL) was added DMAP (3.05 g, 0.025 mol), followed by EDCI (3.88 g, 0.025 mol). The resulting mixture was stirred overnight at room temperature and then washed with 1N HCl solution (500 mL) and water (500 mL). The organic layer was dried over MgSO4, evaporated to dryness, and purified by silica gel column chromatography using 0-10% MeOH in DCM as the eluent. Fractions containing the desired compound were pooled and evaporated to give 9d (6.2 g, 62%) as a white solid. 1 H-NMR (300 MHz, d-chloroform): δ 4.86 (t, 1H), 2.28-2.34 (m, 4H), 1.58-1.63 (m, 8H), 1.26-1.32 (m, 27H), 0.85-0.87 (t, 6H).

[0283] Synthesis of lipid 8 [ka] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(pentadecan-7-yl)nonanedioate (Lipid 8). To a stirred solution of disulfide 13 (synthesis described in Example 6) (250 mg, 0.27 mmol) and acid 9d (120 mg, 0.33 mmol) in DCM (20 ml) was added DMAP (40 mg, 0.33 mmol), followed by EDCI (51 mg, 0.33 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 8 (170 mg, 40%). 1 H-NMR (300MHz, d-chloroform): δ7.29(d, 4H), 7.04(d, 4H), 5.29-5.34(m, 2H), 4.86(m, 1H), 4.11(t, 4H), 3.58(t, 4H), 2.80-2.93(m, 8H), 2.51-2.68(m, 8H), 2.28(m, 2H), 1.97-2.05(m, 8H), 1.70-1.80(m, 4H), 1.50-1.70(m, 18H), 1.10-1.40(m, 58H), 0.87(t, 9H).

[0284] Example 9: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(undecan-3-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (Lipid 9) Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-nonylnonanedioate (14). [ka] Synthesis of 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-hydroxyphenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)-2-oxoethyl)phenyl)9-nonylnonandioate (14). To a stirred solution of disulfide 7 (stepwise synthesis described in Example 1) (3.1 g, 4.8 mmol) and 9-(nonyloxy)-9-oxononanoic acid (9) (synthesis described in Example 1) (1.51 g, 4.8 mmol) in dichloromethane (200 ml) was added DMAP (587 mg, 4.8 mmol) followed by EDCI (746 mg, 4.8 mmol). The resulting mixture was stirred at room temperature overnight, and then saturated sodium bicarbonate solution (50 ml) was added. The reaction mixture was extracted with dichloromethane (2 x 50 ml). The combined organic phases were washed with brine (30 ml), dried over NaSO, and concentrated. The residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent to give the desired product 14 (2.47 g, 55%). The crude product was used directly in the next step without further characterization.

[0285] Synthesis of lipid 9 [ka] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(undecan-3-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (Lipid 9). To a stirred solution of disulfide 14 (250 mg, 0.26 mmol) and acid 9b (synthesis described in Example 6) (110 mg, 0.32 mmol) in dichloromethane (20 ml) was added DMAP (46 mg, 0.37 mmol) followed by EDCI (50 mg, 0.32 mmol). The resulting mixture was stirred at room temperature overnight, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 9 (230 mg, 68%). 1 H-NMR (300MHz, d-chloroform): δ7.28 (d, 4H), 7.04 (d, 4H), 4.86 (m, 1H), 4.06-4.12 (t, 4H), 4.04 (t, 2H), 3.59 (s, 4) H), 2.60-2.90(m, 8H), 2.27-2.60(m, 10H), 1.97(t, 3H), 1.52-1.80(m, 18H), 1.10-1.40(m, 40H), 0.88(t, 9H).

[0286] Example 10: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(tridecan-5-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (Lipid 10) [ka] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(tridecan-5-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (lipid 10). To a stirred solution of disulfide 14 (synthesis described in Example 9) (330 mg, 0.35 mmol) and acid 9c (synthesis described in Example 7) (143 mg, 0.39 mmol) in dichloromethane (20 ml) was added DMAP (47 mg, 0.39 mmol), followed by EDCI (60 mg, 0.39 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over NaSO. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% methanol in dichloromethane as the eluent. The fractions containing the desired compound were evaporated to give lipid 10 (150 mg, 33%). 1 H-NMR (300MHz, d-chloroform): δ7.26(d, 4H), 7.03(d, 4H), 4.86(m, 1H), 4.05-4.11(t, 6H), 3.58(s, 4H), 2.80-2.90(m, 8H), 2.50-2.70(m, 8H), 2.26-2.29(m, 4H), 1.92-1.99(m, 4H), 1.50-1.80(m, 24H), 1.16-1.40(m, 46H), 0.87(t, 9H).

[0287] Example 11: Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(pentadecan-7-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (Lipid 11) [ka] Synthesis of 1-nonyl 9-(4-(2-oxo-2-(2-(1-(2-((2-(4-(2-(2-(4-((9-oxo-9-(pentadecan-7-yloxy)nonanoyl)oxy)phenyl)acetoxy)ethyl)piperidin-1-yl)ethyl)disulfanayl)ethyl)piperidin-4-yl)ethoxy)ethyl)phenyl)nonanedioate (lipid 11). To a stirred solution of disulfide 14 (synthesis described in Example 9) (260 mg, 0.28 mmol) and acid 9d (synthesis described in Example 8) (122 mg, 0.3 mmol) in DCM (20 ml) was added DMAP (37 mg, 0.3 mmol), followed by EDCI (47 mg, 0.3 mmol). The resulting mixture was stirred overnight at room temperature, then washed with saturated sodium bicarbonate solution (20 ml), brine (20 ml), and dried over Na2SO4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography using 0-5% MeOH in DCM as the eluent. The fractions containing the desired compound were evaporated to give lipid 11 (110 mg, 30%). 1 H-NMR (300MHz, d-chloroform): δ7.26 (d, 4H), 7.02 (d, 4H), 4.86 (m, 1H), 4.05-4.11 (t, 6H), 3.59 (s, 4H), 2.80-2.90 (m, 8H), 2.50-2.70(m, 8H), 2.27-2.29(m, 4H), 1.90-2.20(t, 4H), 1.50-1.82(m, 24H), 1.10-1.40(m, 50H), 0.87(t, 9H).

[0288] The following lipids 12-20 in Table 4 were prepared following similar procedures with appropriate starting materials and other modifications within the knowledge of one of ordinary skill in the art. [Table 4-1] [Table 4-2]

[0289] Example 2: Preparation of lipid nanoparticle formulations Lipid nanoparticles (LNPs) were prepared at a total lipid to ceDNA weight ratio of approximately 10:1 to 30:1. Briefly, ionizable lipids of the present invention, non-cationic lipids (e.g., distearoylphosphatidylcholine (DSPC)), components for membrane fusogenicity (e.g., sterols, e.g., cholesterol), and conjugated lipid molecules (e.g., PEG-lipids, e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol, with an average PEG molecular weight of 2000 ("PEG-DMG")) were solubilized in alcohol (e.g., ethanol) at molar ratios of, for example, 50:10:37:3 or 20:40:38:2. ceDNA was diluted to the desired concentration with a buffer solution. For example, ceDNA was diluted to a concentration of 0.1 mg / ml to 0.25 mg / ml in a buffer solution containing sodium acetate, sodium acetate and magnesium chloride, citric acid, malic acid, or malic acid and sodium chloride. In one example, ceDNA was diluted to 0.2 mg / mL in 10–50 mM citrate buffer (pH 4). The alcoholic lipid solution was mixed with the aqueous ceDNA solution at a ratio of approximately 1:5–1:3 (vol / vol) using, for example, a syringe pump or impingement jet mixer, with a total flow rate exceeding 10 mL / min. In one example, the alcoholic lipid solution was mixed with the aqueous ceDNA at a ratio of approximately 1:3 (vol / vol) at a flow rate of 12 mL / min. The alcohol was removed and the buffer replaced with PBS by dialysis. Alternatively, the buffer was replaced with PBS using centrifuge tubes. Alcohol removal and simultaneous buffer exchange were achieved, for example, by dialysis or tangential flow filtration. The resulting lipid nanoparticles were filtered through a 0.2 μm pore sterile filter.

[0290] In one study, exemplary ceDNA-containing lipid nanoparticles were prepared using a lipid solution containing SS-OP, DSPC, cholesterol, and DMG-PEG2000 (molar ratio 50:10:37:3). In some cases, a tissue-targeting moiety such as N-acetylgalactosamine (GalNAc) was included. GalNAc moieties, such as triple-armed GalNAc (GalNAc3) or quadruple-armed GalNAc (GalNAc4), can be synthesized as known in the art (see WO2017 / 084987 and WO2013 / 166121) and chemically conjugated to lipids or PEG as known in the art (see Resen et al., J. Biol. Chem. (2001) "Determination of the Upper Size Limit for Uptake and Processing of Ligands by the Asialoglycoprotein Receptor on Hepatocytes in Vitro and in Vivo" 276:375577-37584). An aqueous solution of ceDNA in a buffer solution was prepared. The lipid and ceDNA solutions were mixed using an in-house procedure on a NanoAssembler at a lipid-to-ceDNA ratio of 1:3 (v / v) and a total flow rate of 12 mL / min. [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0291] Species (number, sex, age): CD-1 mice (N=65 and 5 spares, male, approximately 4 weeks old on arrival).

[0292] Cage-side observations: Cage-side observations were performed daily.

[0293] Clinical Observations: Clinical observations were performed at approximately 1, approximately 5 to 6, and approximately 24 hours after test article administration on Day 0. Additional observations were performed on each exception. Body weights of all animals were recorded on Days 0, 1, 2, 3, 4, and 7 (prior to euthanasia), if applicable. Additional body weights were recorded as needed.

[0294] Dose Administration: Test article (LNP:ceDNA-Luc) was administered at 5 mL / kg on day 0 to groups 1-38 via intravenous administration into the lateral tail vein.

[0295] In-life imaging: On day 4, all animals were administered luciferin at 150 mg / kg (60 mg / mL) via intraperitoneal (IP) injection at 2.5 mL / kg. Within 15 minutes after each luciferin administration, all animals underwent an IVIS imaging session according to the in vivo imaging protocol described below.

[0296] Anesthesia Recovery: Animals were continuously monitored while under anesthesia, during recovery, and until ambulation.

[0297] Intermediate blood sampling: All animals underwent interim blood sampling on day 0, 5 to 6 hours (5.0 hours or more, 6.5 hours or less) after administration of the test substance.

[0298] After collection, animals received 0.5–1.0 mL of lactated Ringer's solution subcutaneously.

[0299] Whole blood for serum was collected by tail vein nick, saphenous vein, or orbital sinus puncture (under inhaled isoflurane). Whole blood was collected in a serum separator equipped with a clot activator tube and processed into one (1) serum aliquot.

[0300] In vivo imaging protocol Luciferin stock powder was stored at -20°C according to labeling. • Formulated luciferin was stored in 1 mL aliquots at 2-8°C, protected from light. • Formulated luciferin was stable for up to 3 weeks at 2-8°C, protected from light, and for approximately 12 hours at room temperature (RT). Luciferin was dissolved in a sufficient volume of PBS to a target concentration of 60 mg / mL, and adjusted to pH=7.4 with 5 M NaOH (approximately 0.5 μl / mg luciferin) and HCl (approximately 0.5 μL / mg luciferin) as needed. • The appropriate volume was prepared according to protocol, including at least approximately 50% overage.

[0301] Injection and Imaging (Note: Up to 5 animals may be imaged at one time) • Shaved the animal's coat (if necessary). ● 150 mg / kg luciferin in 60 mg / mL PBS was injected via intraperitoneal cavity according to the protocol. ●Imaging was performed immediately after administration or up to 15 minutes after administration. To anesthetize the animals during the imaging session, the isoflurane vaporizer was set to 1-3% (usually at 2.5%). Isoflurane anesthesia for imaging sessions: The animal was placed in the isoflurane chamber and allowed approximately 2-3 minutes for the isoflurane to take effect. ○Make sure the anesthesia level on the side of the IVIS device is in the "on" position. ○The animal was placed in the IVIS machine

[0302] The desired acquisition protocol was performed at the highest sensitivity setting.

[0303] result Research A As shown in Figure 1, on day 4, groups of mice treated with ceDNA-luciferase (ceDNA-luc) formulated with lipid 1, lipid 2, lipid 3, or lipid 5 (LNPs 5, 4, 6, and 3, respectively, in Figure 1) showed equal or higher luciferase expression and / or activity compared to groups treated with the positive control ceDNA LNPs used in Study A (LNPs 1, 2, and 7-12, each of which was ceDNA-luc formulated with SS-OP lipid), demonstrating that the ionizable lipids described herein have superior physical attributes as lipid nanoparticle delivery vehicles.

[0304] Research B As shown in Figure 2, consistent with the observations in Figure 1 of Study A above, on day 4, the groups of mice treated with ceDNA-luc formulated with lipid 1, lipid 2, lipid 3, or lipid 5 (LNPs 17, 16, 18, and 15, respectively, in Figure 2) exhibited equal or higher luciferase activity compared to the group treated with the positive control ceDNA LNP used in Study B (LNP 13, which was ceDNA-luc formulated with SS-OP lipid), demonstrating that the ionizable lipids described herein have superior physical attributes as lipid nanoparticle delivery vehicles.

[0305] Research C Lipids 1 and 3, which showed the highest luciferase expression and / or activity in Studies A and B, were further studied in Study C for dose response. As shown in Figure 3, consistent with the observations in Figures 1 and 2 from Studies A and B, on day 4, groups of mice treated with ceDNA-luc formulated with lipid 1 or lipid 3 (LNP20 and 21, respectively, in Figure 1) showed higher luciferase expression and / or activity at both approximately 25 mg / kg and 1 mg / kg compared to groups treated with the positive control ceDNA LNP used in Study C (LNP19, which was ceDNA-luc formulated with SS-OP lipid), demonstrating that the ionizable lipids described herein have superior physical attributes as lipid nanoparticle delivery vehicles. Furthermore, the results in Figure 3 show that LNP20, when increased in dose from 0.25 mg / kg to 1 mg / kg, showed a greater increase in luciferase expression and / or activity compared to LNP19, also tested at the same two dose levels. These results indicate that LNPs formulated with ionizable lipids of the present disclosure are more responsive to different dosage levels, and that the expression level of the transgene insert in ceDNA encapsulated by LNPs formulated with ionizable lipids of the present disclosure can be more easily adjusted to the level required to exert its therapeutic effect for a particular disease, thereby demonstrating another desirable technical feature of these ionizable lipids as lipid nanoparticle delivery vehicles.

[0306] Research D In Study D, LNPs formulated with lipid 6, lipid 7, and lipid 8 (LNPs 24, 25, and 26 in Figures 4A and 4B, respectively) and ceDNA-luc were evaluated for luciferase expression and / or activity in mice and compared to LNPs formulated with ionizable lipid A and SS-OP lipids (LNPs 22 and 23 in Figures 4A and 4B) and ceDNA-luc. As shown in Figure 4A, on day 4, the groups of mice treated with ceDNA-luc constructs formulated with lipid 6, lipid 7, and lipid 8 showed equal or higher luciferase expression and / or activity compared to the group treated with ceDNA-luc formulated with SS-OP lipid (i.e., LNP23). Figure 4B shows that the ceDNA-luc constructs formulated with lipid 6, lipid 7, and lipid 8 were also well tolerated in mice, as treatment did not cause any changes in body weight in the mice on day 1. In contrast, as seen in Figure 4B, mice treated with ceDNA-luc formulated with ionizable lipid A (i.e., LNP22) suffered significant weight loss on day 1, thereby indicating that the lipid was not well tolerated by the animals.

[0307] Research E In Study E, LNPs formulated with lipid 9, lipid 10, and lipid 11 (LNP28, 29, and 30 in Figures 5A and 5B, respectively) and ceDNA-luc were evaluated for luciferase expression and / or activity in mice and compared to LNPs formulated with SS-OP lipid (LNP27 in Figures 5A and 5B) and ceDNA-luc. As shown in Figure 5A, on day 4, the groups of mice treated with ceDNA-luc constructs formulated with lipid 9, lipid 10, and lipid 11 exhibited equal or higher luciferase expression and / or activity compared to the group treated with ceDNA-luc formulated with SS-OP lipid (i.e., LNP27). Figure 5B shows that, with the exception of the outlier data point in LNP30, the ceDNA-luc constructs formulated with lipid 9, lipid 10, and lipid 11 were generally well tolerated in mice, as treatment did not cause significant changes in body weight in mice on day 1.

[0308] Thus, Studies A-E collectively demonstrate that LNPs formulated with the ionizable lipids of the present disclosure (i) have excellent in vivo expression levels of ceDNA transgene inserts, (ii) are responsive to different dosage levels, thereby allowing the in vivo expression levels of ceDNA transgene inserts to be adjusted as needed, and (iii) are well tolerated in vivo.

[0309] References All publications and references cited in this specification and the examples herein, including but not limited to patents and patent applications, are incorporated by reference in their entirety as if each individual publication or reference was specifically and individually indicated to be incorporated by reference herein as if fully set forth. Any patent application to which this application claims priority is also incorporated by reference herein in the manner described above for publications and references. The present invention provides, for example, the following items. (Item 1) A lipid having the formula (I): [ka] During the ceremony, a is an integer ranging from 1 to 20; b is an integer ranging from 2 to 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. (Item 2) The lipid is of formula (II), [ka] 2. The lipid according to item 1, or a pharmaceutically acceptable salt thereof, wherein c and d are each independently an integer ranging from 1 to 8. (Item 3) 3. The lipid according to item 2, or a pharmaceutically acceptable salt thereof, wherein c and d are each independently an integer ranging from 2 to 8. (Item 4) 4. The lipid according to item 2 or 3, or a pharmaceutically acceptable salt thereof, wherein c and d are each independently an integer in the range of 4 to 8. (Item 5) 5. The lipid according to any one of items 2 to 4, or a pharmaceutically acceptable salt thereof, wherein c and d are each independently an integer in the range of 6 to 8. (Item 6) 3. The lipid according to item 2, or a pharmaceutically acceptable salt thereof, wherein c and d are each independently 1, 3, 5, or 7. (Item 7) 7. The lipid according to any one of items 2 to 6, or a pharmaceutically acceptable salt thereof, wherein at least one of c and d is 7. (Item 8) The lipid according to any one of items 1 to 7, wherein the lipid is of formula (III): [ka] or a pharmaceutically acceptable salt thereof. (Item 9) 9. The lipid according to any one of items 1 to 8, or a pharmaceutically acceptable salt thereof, wherein b is an integer ranging from 3 to 9. (Item 10) 10. The lipid according to any one of items 1 to 9, or a pharmaceutically acceptable salt thereof, wherein b is an integer ranging from 5 to 7. (Item 11) 11. The lipid according to any one of items 1 to 10, wherein b is 5 or 7, or a pharmaceutically acceptable salt thereof. (Item 12) 12. The lipid according to any one of items 1 to 11, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 2 to 18. (Item 13) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 3 to 17. (Item 14) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 6 to 18. (Item 15) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 4 to 12. (Item 16) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 2 to 5. (Item 17) 17. The lipid according to item 16, or a pharmaceutically acceptable salt thereof, wherein a is 3. (Item 18) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 6 to 8. (Item 19) 19. The lipid according to item 18, or a pharmaceutically acceptable salt thereof, wherein a is 7. (Item 20) 19. The lipid according to item 18, or a pharmaceutically acceptable salt thereof, wherein a is 8. (Item 21) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 16 to 18. (Item 22) 22. The lipid according to item 21, or a pharmaceutically acceptable salt thereof, wherein a is 17. (Item 23) 13. The lipid according to any one of items 1 to 12, or a pharmaceutically acceptable salt thereof, wherein a is an integer ranging from 9 to 11. (Item 24) 24. The lipid according to item 23, or a pharmaceutically acceptable salt thereof, wherein a is 10. (Item 25) R 1 However, it is absent or (C5~C 15 ) alkenyl, -C(O)O(C4-C 18 ) alkyl, and (C4-C 16 25. The lipid according to any one of items 1 to 24, or a pharmaceutically acceptable salt thereof, wherein the lipid is selected from the group consisting of cyclopropyl substituted with alkyl. (Item 26) R 1 However, it is absent or (C5~C 12 ) alkenyl, -C(O)O(C4-C 12 ) alkyl, and (C4-C 12 26. The lipid according to any one of items 1 to 25, or a pharmaceutically acceptable salt thereof, wherein the lipid is selected from cyclopropyl substituted with alkyl. (Item 27) R 1 However, it is absent or (C5~C 10 ) alkenyl, -C(O)O(C4-C 10 ) alkyl, and (C4-C 10 27. The lipid according to any one of items 1 to 26, or a pharmaceutically acceptable salt thereof, wherein the lipid is selected from cyclopropyl substituted with alkyl. (Item 28) R 1 But C 10 28. The compound according to any one of items 1 to 27, or a pharmaceutically acceptable salt thereof, which is alkenyl. (Item 29) R 1 -C(O)O(C-C 20 ) alkyl, -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 28. The lipid according to any one of items 1 to 27, wherein the alkyl in the alkyl is an unbranched alkyl, or a pharmaceutically acceptable salt thereof. (Item 30) R 1 30. The lipid according to item 29, or a pharmaceutically acceptable salt thereof, wherein is —C(O)O(C alkyl). (Item 31) -C(O)O(C4-C 18 ) alkyl, -C(O)O(C4-C 12 ) alkyl, or -C(O)O(C4-C 10 28. The lipid according to any one of items 25 to 27, wherein the alkyl in the alkyl is a branched alkyl, or a pharmaceutically acceptable salt thereof. (Item 32) R 1 But -C(O)O(C 17 32. The lipid according to item 31, or a pharmaceutically acceptable salt thereof, wherein the lipid is hydroxypropyl or hydroxypropyl. (Item 33) R 1 is selected from any group listed in Table 1, or a pharmaceutically acceptable salt thereof. (Item 34) R 2 is selected from any group listed in Table 2, or a pharmaceutically acceptable salt thereof. (Item 35) 2. The lipid according to item 1, or a pharmaceutically acceptable salt thereof, wherein the lipid is selected from any lipid listed in Table 3. (Item 36) A lipid nanoparticle (LNP) comprising the lipid according to any one of items 1 to 35, or a pharmaceutically acceptable salt thereof, and a nucleic acid. (Item 37) 37. The lipid nanoparticle according to claim 36, wherein the nucleic acid is encapsulated within the lipid. (Item 38) 38. The lipid nanoparticle of item 36 or 37, wherein the nucleic acid is selected from the group consisting of a minigene, a plasmid, a minicircle, a small interfering RNA (siRNA), a microRNA (miRNA), an antisense oligonucleotide (ASO), a ribozyme, a ceDNA, a ministring, doggybone™, a protelomeric closed-end DNA or a dumbbell linear DNA, a dicer substrate dsRNA, a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a microRNA (miRNA), an mRNA, a tRNA, a rRNA, a DNA viral vector, a viral RNA vector, a non-viral vector, and any combination thereof. (Item 39) 39. The lipid nanoparticle of item 38, wherein the nucleic acid is closed-end DNA (ceDNA). (Item 40) 40. The lipid nanoparticle according to any one of items 36 to 39, further comprising a sterol. (Item 41) 41. The lipid nanoparticles according to item 40, wherein the sterol is cholesterol or beta-sitosterol. (Item 42) 42. The lipid nanoparticle according to any one of items 36 to 41, further comprising a PEG-lipid conjugate. (Item 43) 43. The lipid nanoparticle of item 42, wherein the PEG-lipid conjugate is 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG). (Item 44) The lipid nanoparticles according to any one of Items 36 to 43, further comprising a non-cationic lipid. (Item 45) The non-cationic lipid is selected from the group consisting of distearoyl-sn-glycero-phosphoethanolamine, 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 (e.g., 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (e.g., 16-O-dimethyl PE), 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (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 oleoyl phosphatidylglycerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,Item 45. The lipid nanoparticle according to item 44, wherein the lipid nanoparticle 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, phosphatidicaside, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. (Item 46) 46. ​​The lipid nanoparticle according to item 45, wherein the non-cationic lipid is selected from the group consisting of dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dioleoyl-phosphatidylethanolamine (DOPE). (Item 47) 47. The lipid nanoparticle of item 46, wherein the PEG-lipid conjugate is present at a molar percentage of about 1.5% to about 4%. (Item 48) 48. The lipid nanoparticle of item 47, wherein the PEG-lipid conjugate is present at a molar percentage of about 2% to about 3%. (Item 49) Item 49. The lipid nanoparticle of item 48, wherein the PEG-lipid conjugate is present at a molar percentage of about 2.5% to about 3%. (Item 50) 50. The lipid nanoparticle of item 49, wherein the PEG-lipid conjugate is present at a molar percentage of about 3%. (Item 51) 51. The lipid nanoparticle according to any one of items 42 to 50, wherein the PEG-lipid conjugate is DMG-PEG. (Item 52) 52. The lipid nanoparticle according to any one of items 36 to 51, wherein the cholesterol or beta-sitosterol is present in a molar percentage of about 20% to about 40% and the lipid is present in a molar percentage of about 80% to about 60%. (Item 53) 53. The lipid nanoparticle of item 52, wherein the cholesterol or beta-sitosterol is present in a molar percentage of about 40% and the lipid is present in a molar percentage of about 50%. (Item 54) 40. The lipid nanoparticle according to any one of items 36 to 39, further comprising cholesterol, a PEG-lipid conjugate, and a non-cationic lipid. (Item 55) 55. The lipid nanoparticle according to item 54, wherein the PEG-lipid conjugate is present at about 1.5% to about 4%. (Item 56) 56. The lipid nanoparticle according to item 55, wherein the PEG-lipid conjugate is present at about 2% to about 3%. (Item 57) 57. The lipid nanoparticle according to item 56, wherein the PEG-lipid conjugate is present at about 2.5% to about 3%. (Item 58) 58. The lipid nanoparticle of item 57, wherein the PEG-lipid conjugate is present at about 3%. (Item 59) 48. The lipid nanoparticle according to any one of items 42 to 47, wherein the cholesterol is present in a molar percentage of about 30% to about 50%. (Item 60) 61. The lipid nanoparticle according to any one of items 54 to 60, wherein the PEG-lipid conjugate is DMG-PEG2000. (Item 61) 61. The lipid nanoparticle of any one of items 53 to 60, wherein the lipid is present in a molar percentage of about 42.5% to about 62.5%. (Item 62) 61. The lipid nanoparticle of any one of items 53 to 60, wherein the non-cationic lipid is present in a molar percentage of about 2.5% to about 12.5%. (Item 63) 61. The lipid nanoparticle of any one of items 53 to 60, wherein the cholesterol is present in a molar percentage of about 40%, the lipid is present in a molar percentage of about 52.5%, the non-cationic lipid is present in a molar percentage of about 7.5%, and the PEG-lipid conjugate is present in a molar percentage of about 3%. (Item 64) 64. The lipid nanoparticle according to any one of items 36 to 63, further comprising dexamethasone palmitate. (Item 65) 65. The lipid nanoparticle according to any one of items 36 to 64, wherein the nanoparticle has a diameter in the range of about 50 nm to about 110 nm. (Item 66) 65. The lipid nanoparticle of any one of items 36 to 64, wherein the nanoparticles are less than about 100 nm in size. (Item 67) 67. The lipid nanoparticle of item 66, wherein the particles are less than about 70 nm in size. (Item 68) 68. The lipid nanoparticle of item 67, wherein the particles are less than about 60 nm in size. (Item 69) 40. The lipid nanoparticle of item 39, wherein the particles have a total lipid to ceDNA ratio of about 10:1. (Item 70) 40. The lipid nanoparticle of item 39, wherein the particles have a total lipid to ceDNA ratio of about 20:1. (Item 71) 40. The lipid nanoparticle of item 39, wherein the particles have a total lipid to ceDNA ratio of about 30:1. (Item 72) 40:1 39. The lipid nanoparticle of claim 39, wherein the particle has a total lipid to ceDNA ratio of about 40:1. (Item 73) 73. The lipid nanoparticle of any one of items 36 to 72, further comprising a tissue-specific targeting moiety. (Item 74) 74. The lipid nanoparticle of item 73, wherein the tissue-specific targeting moiety is N-acetylgalactosamine (GalNAc), wherein GalNAc is conjugated to the PEG-lipid conjugate, and wherein the GalNAc-conjugated PEG-lipid conjugate is present in the particle at a molar percentage of about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%. (Item 75) 75. The lipid nanoparticle of item 74, wherein the GalNac-linked PEG-lipid conjugate is present in the particle at a molar percentage of about 0.5%. (Item 76) 76. The lipid nanoparticle according to any one of items 36 to 75, further comprising about 10 mM to about 30 mM malic acid. (Item 77) 77. The lipid nanoparticles according to item 76, comprising about 20 mM malic acid. (Item 78) 78. The lipid nanoparticle according to any one of items 36 to 77, further comprising about 30 mM to about 50 mM NaCl. (Item 79) 79. The lipid nanoparticles according to item 78, further comprising about 40 mM NaCl. (Item 80) 80. The lipid nanoparticle of any one of items 36 to 79, further comprising about 20 mM to about 100 mM MgCl. (Item 81) 82. The lipid nanoparticle according to claim 39, wherein the ceDNA is a closed-end linear double-stranded DNA. 40. The lipid nanoparticle of item 39, wherein the ceDNA comprises an expression cassette, the expression cassette comprising a promoter sequence and a transgene. (Item 83) 83. The lipid nanoparticle of item 82, wherein the expression cassette comprises a polyadenylation sequence. (Item 84) 84. The lipid nanoparticle of any one of items 81 to 83, wherein the ceDNA comprises at least one inverted terminal repeat (ITR) adjacent to either the 5' or 3' end of the expression cassette. (Item 85) 85. The lipid nanoparticle of item 84, wherein the expression cassette is flanked by two ITRs, the two ITRs comprising one 5' ITR and one 3' ITR. (Item 86) 85. The lipid nanoparticle of item 84, wherein the expression cassette is linked to the ITR at the 3' end (3'ITR). (Item 87) 85. The lipid nanoparticle of item 84, wherein the expression cassette is linked to the ITR at the 5' end (5'ITR). (Item 88) 85. The lipid nanoparticle of item 84, wherein at least one of the 5' ITR and 3' ITR is a wild-type AAV ITR. (Item 89) 85. The lipid nanoparticle of item 84, wherein at least one of the 5'ITR and 3'ITR is a modified ITR. (Item 90) 85. The lipid nanoparticle of item 84, wherein the ceDNA further comprises a spacer sequence between the 5' ITR and the expression cassette. (Item 91) 85. The lipid nanoparticle of item 84, wherein the ceDNA further comprises a spacer sequence between the 3' ITR and the expression cassette. (Item 92) 92. The lipid nanoparticle of item 90 or 91, wherein the spacer sequence is at least 5 base pairs long. (Item 93) 93. The lipid nanoparticle according to item 92, wherein the spacer sequence is 5 to 100 base pairs in length. (Item 94) 93. The lipid nanoparticle according to item 92, wherein the spacer sequence is 5 to 500 base pairs in length. (Item 95) 95. The lipid nanoparticle according to any one of items 38 to 94, wherein the ceDNA has a nick or a gap. (Item 96) 85. The lipid nanoparticle according to item 84, wherein the ITR is an ITR derived from an AAV serotype, derived from a goose virus ITR, derived from a B19 virus ITR, or a wild-type ITR from a parvovirus. (Item 97) 97. The lipid nanoparticle of item 96, wherein the AAV serotype is selected from the group comprising AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and AAV12. (Item 98) 85. The lipid nanoparticle of item 84, wherein the ITR is a mutant ITR and the ceDNA optionally comprises an additional ITR that is different from the first ITR. (Item 99) 85. The lipid nanoparticle of claim 84, wherein the ceDNA comprises two mutant ITRs at both the 5' and 3' ends of the expression cassette, and optionally the two mutant ITRs are symmetric mutants. (Item 100) 40. The lipid nanoparticle of item 39, wherein the ceDNA is a CELiD, a DNA-based minicircle, a MIDGE, a ministering DNA, a dumbbell-shaped linear double-stranded closed-end DNA containing two hairpin structures of ITRs at the 5' and 3' ends of the expression cassette, or doggybone™ DNA. (Item 101) A pharmaceutical composition comprising the lipid nanoparticles according to any one of items 36 to 100 and a pharmaceutically acceptable excipient. (Item 102) 26. A pharmaceutical composition comprising the lipid according to any one of items 1 to 25, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient. (Item 103) A method for treating a genetic disorder in a subject, the method comprising administering to the subject an effective amount of the lipid nanoparticles according to any one of items 36 to 100, or an effective amount of the pharmaceutical composition according to item 101. (Item 104) Item 104. The method of item 103, wherein the subject is a human. (Item 105) The genetic 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, sickle cell anemia, thalassemia, xeroderma pigmentosum, Fanconi anemia, retinitis pigmentosa, ataxia-telangiectasia, Bloom's syndrome, retinoblastoma, mucopolysaccharidosis (e.g., Hurler syndrome (MPS type I), Scheie syndrome (MPS type IS), Hurler-Scheie syndrome (MPS type I HS), Hunter syndrome (MPS type II), Sanfilippo syndrome types A, B, C, and D (MPS III), A, B, C, and D), Morquio A and B (MPS IVA and MPS IVB), Marote-Lamy syndrome (MPS VI), Sly syndrome (MPS VII), hyaluronidase deficiency (MPSIX), Niemann-Pick disease 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, Fabry disease, 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, 105. The method of item 103 or 104, wherein the patient is selected from the group consisting of 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, generalized arterial calcification of infancy (GACI), Leber's congenital amaurosis, Stargardt's macular dystrophy (ABCA4), ornithine transcarbamylase (OTC) deficiency, Usher syndrome, 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. (Item 106) 106. The method of claim 105, wherein the genetic disorder is Leber congenital amaurosis (LCA). (Item 107) Item 107. The method of item 106, wherein the LCA is LCA10. (Item 108) Item 106. The method of item 105, wherein the genetic disorder is Niemann-Pick disease. (Item 109) 106. The method of claim 105, wherein the genetic disorder is Stargardt's macular dystrophy. (Item 110) 106. The method of item 105, wherein the genetic disorder is glucose-6-phosphatase (G6Pase) deficiency (glycogen storage disease type I) or Pompe disease (glycogen storage disease type II). (Item 111) 106. The method of claim 105, wherein the genetic disorder is hemophilia A (factor VIII deficiency). (Item 112) Item 113. The method of Item 105, wherein the genetic disorder is hemophilia B (factor IX deficiency). 106. The method of claim 105, wherein the genetic disorder is Hunter syndrome (mucopolysaccharidosis type II). (Item 114) 106. The method of claim 105, wherein the genetic disorder is cystic fibrosis. (Item 115) Item 106. The method of item 105, wherein the genetic disorder is dystrophic epidermolysis bullosa (DEB). (Item 116) Item 117. The method of item 105, wherein the genetic disorder is phenylketonuria (PKU). 106. The method of item 105, wherein the genetic disorder is progressive familial intrahepatic cholestasis (PFIC). (Item 118) 106. The method of claim 105, wherein the genetic disorder is Wilson's disease. (Item 119) 106. The method of item 105, wherein the genetic disorder is Gaucher disease type I, II, or III.

Claims

1. A composition for treating a disease or disorder in a subject requiring treatment of a disease or disorder, The composition comprises lipid nanoparticles (LNPs), The aforementioned LNP is a nucleic acid and formula (I): 【Chemistry 1】 A lipid represented by, or a pharmaceutically acceptable salt thereof, wherein, a is an integer in the range of 1 to 20. b is an integer in the range of 2 to 10. R1 is selected from the group consisting of (C2-C20) alkenyl, -C(O)O(C2-C20) alkyl, and cyclopropyl substituted with (C2-C20) alkyl. R2 is an (C2-C20) alkyl group. composition.

2. The lipid is of formula (II): 【Transformation 34】 The composition according to claim 1, which is represented by or a pharmaceutically acceptable salt thereof, wherein c and d are each independently integers in the range of 1 to 8.

3. The composition according to claim 2, wherein c and d are each independently integers in the range of 2 to 8, 4 to 8, or 6 to 8.

4. The composition according to claim 2, wherein c and d are each independently 1, 3, 5, or 7.

5. The composition according to any one of claims 2 to 4, wherein at least one of c and d is 7.

6. The lipid is of formula (III): 【Chemistry 35】 The composition according to any one of claims 1 to 5, which is represented by or a pharmaceutically acceptable salt thereof.

7. The composition according to any one of claims 1 to 6, wherein b is an integer in the range of 3 to 9 or 5 to 7.

8. The composition according to any one of claims 1 to 7, wherein b is 5 or 7.

9. The composition according to any one of claims 1 to 8, wherein a is an integer in the range of 2 to 18, or 3 to 17, or 6 to 18, or 4 to 12, or 2 to 5, or 6 to 8, or 16 to 18, or 9 to 11.

10. The composition according to claim 9, wherein a is 3, 7, 8, 10, or 17.

11. The composition according to any one of claims 1 to 10, wherein R1 is selected from the group consisting of (C5 to C15) alkenyl, -C(O)O(C4 to C18) alkyl, and (C4 to C16) alkyl-substituted cyclopropyl.

12. The composition according to any one of claims 1 to 11, wherein R1 is selected from the group consisting of (C5-C12) alkenyl, -C(O)O(C4-C12) alkyl, and (C4-C12) alkyl-substituted cyclopropyl.

13. The composition according to any one of claims 1 to 12, wherein R1 is selected from the group consisting of (C5-C10) alkenyl, -C(O)O(C4-C10) alkyl, and (C4-C10) alkyl-substituted cyclopropyl.

14. The composition according to any one of claims 1 to 13, wherein R1 is a C10 alkenyl.

15. The composition according to any one of claims 1 to 13, wherein R1 is a -C(O)O(C2-C20) alkyl, -C(O)O(C4-C18) alkyl, -C(O)O(C4-C12) alkyl, or -C(O)O(C4-C10) alkyl, where the alkyl is an unbranched alkyl.

16. The composition according to claim 15, wherein R1 is -C(O)O(C9 alkyl).

17. The composition according to any one of claims 11 to 13, wherein R1 is a -C(O)O(C4-C18) alkyl, a -C(O)O(C4-C12) alkyl, or a -C(O)O(C4-C10) alkyl, where the alkyl is a branched alkyl.

18. The composition according to claim 17, wherein R1 is -C(O)O(C17 alkyl).

19. R1 is, 【Transformation 36】 Selected from the group consisting of and / or R 2, 【Chemistry 37】 A composition according to any one of claims 1 to 10, selected from the group consisting of the following.

20. The lipid is 【Transformation 38】 【Chemistry 39】 【Chemistry 40】 【Chemistry 41】 【Chemistry 42】 【Chemistry 43】 【Chemistry 44】 【Chemistry 45】 The composition according to claim 1, which is selected from the group consisting of or a pharmaceutically acceptable salt thereof.

21. The lipid is lipid 7: 【Chemistry 101】 The composition according to claim 20, which is 1-(4-(2-(2-(1-(2-((2-(4-(2-(2-(4-(oleoyloxy)phenyl)acetoxy)ethyl)piperidine-1-yl)ethyl)disulfaneyl)ethyl)piperidine-4-yl)ethoxy)-2-oxoethyl)phenyl)9-(tridecane-5-yl)nonanediate, or a pharmaceutically acceptable salt thereof.

22. The composition according to any one of claims 1 to 21, wherein the nucleic acid contained in the lipid nanoparticles is selected from the group consisting of minigenes, plasmids, minicircles, small interfering RNA (siRNA), antisense oligonucleotides (ASOs), ribozymes, ceDNA, ministrings, doggybone™, protelomere closed-end DNA, dumbbell linear DNA, Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, DNA viral vectors, viral RNA vectors, nonviral vectors, and any combination thereof.

23. The composition according to any one of claims 1 to 22, wherein the lipid nanoparticles further comprise at least one lipid selected from the group consisting of sterols, PEG-lipid conjugates, and noncationic lipids.

24. The composition according to any one of claims 1 to 23, wherein the lipid nanoparticles have a diameter in the range of about 50 nm to about 110 nm, or the lipid nanoparticles have a size of less than about 100 nm in diameter, less than about 70 nm in diameter, or less than about 60 nm in diameter.

25. The composition according to any one of claims 1 to 24, wherein the lipid nanoparticles further comprise a tissue-specific targeting moiety.

26. The composition according to claim 25, wherein the tissue-specific targeting portion is N-acetylgalactosamine (GalNAc) or a peptide.

27. ​​The composition according to claim 26, wherein GalNAc is bound to a second PEG-lipid conjugate to form a GalNAc-bound PEG-lipid conjugate.

28. The composition according to claim 27, wherein the GalNAc-bound PEG-lipid conjugate is present in the lipid nanoparticles in a molar percentage of about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, or about 0.1%.

29. The composition according to any one of claims 1 to 28, wherein the subject is a human.

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